Superoxide dismutase

ABSTRACT

A superoxide dismutase originally found in extracellular body fluids and therefore termed extracellular superoxide dismutase (EC-SOD) is prepared by growing a cell line, preferably of mammalian origin, producing EC-SOD and recovering the EC-SOD secreted from the cells or by inserting a DNA sequence encoding EC-SOD into a suitable vector, introducing the recombinant vector into a host cell, growing the cell and recovering the EC-SOD produced. 
     EC-SOD may be used for the prophylaxis or treatment of diseases or disorders associated with the presence or formation of superoxide radicals.

This is a continuation of application Ser. No. 07/576,114 filed Aug. 27,1990, now U.S. Pat. No. 5,130,245, which is a continuation of Ser. No.06/902,596, filed Sep. 2, 1986 now abandoned, the contents of which arehereby incorporated by reference.

The present invention relates to a superoxide dismutase, methods ofproducing the superoxide dismutase and the use thereof for the purposeof therapeutic treatment.

There is a very strong thermodynamic driving force for the reactionsbetween oxygen and biochemical compounds in the body such as proteins,carbohydrates, lipids and nucleic acids. If such reactions go tocompletion, water, carbon dioxide and a number of waste products areformed as end products concomitantly with the release of large amountsof energy. Oxidation of biological compounds is the source of energy ofliving organisms. Fortunately such reactions occur spantaneously veryslowly due to reaction barriers. These barriers are overcome by theenzymes in intermediary metabolism, and the final reaction with oxygentakes place in the mitochondria, where the oxygen is reduced by fourelectrons to water without the liberation of any intermediate products.The reaction is accomplished by cytochrome oxidase complex in theelectron transport chain and the energy is bound by the formation ofATP.

However, the direct four-step reduction of oxygen to water is almostunique, and when oxygen reacts spontaneously or catalysed by enzymes itis forced to react one step at a time for mechanistic reasons(enzyme-catalysed reactions sometimes require two steps). A series ofreactive and toxic intermediates are formed, namely the superoxideradical (O₂ •⁻) hydrogen peroxide (H₂ O₂), and the hydroxyl radical(OH•) in that order, as shown below: ##STR1##

Two these, O₂ •⁻ and OH•, have single unpaired electrons and aretherefore called free radicals. A few percent of the oxygen consumptionin the body has been estimated to lead to the formation of the toxicreduction intermediates. The toxic effects of oxygen are mainlyascribable to the actions of these intermediates.

Oxygen in itself reacts slowly with most biochemical compounds. Thetoxic reactions are in general initiated by processes giving rise tooxygen radicals, which in themselves cause direct damage to biochemicalcompounds or start chain reactions involving oxygen.

Some compounds react spontaneously with oxygen, i.e. they autoxidize.Virtually all autoxidations result in the formation of toxic oxygenreduction intermediates. Autoxidation of adrenalin, pyrogallol andseveral other compounds leads to the formation of the superoxideradical. The superoxide radical is also released when methemoglobin isformed from oxyhemoglobin. Furthermore, some oxidases form superoxide.The most important of these enzymes is xanthine oxidase, which oxidizeshypoxanthine and xanthine to uric acid. A minor part of the oxygenreduction in mitochondria leads to the formation of superoxide andsubsequently hydrogen peroxide. The microsomal cytochrome P₄₅₀ systemalso releases superoxide. When ionizing radiation passes through anaqueous solution containing oxygen, the superoxide radical is theradical formed in the highest concentration. Upon activation ofphagocytosing leukocytes (polymorphonuclears, monocutes, macrophages,eosinophils) large amounts of superoxide are released. The toxic oxygenreduction products so formed are of fundamental importance for thekilling ability of the cells, but might also lead to damage in thesurrounding tissue.

Hydrogen peroxide is always formed when superoxide is formed by way ofthe dismutation reaction. Most oxidases in the body directly reduceoxygen to hydrogen peroxide.

The most reactive of the intermediates is the hydroxyl radical. It canbe formed when hydrogen peroxide reacts with Fe²⁺ or Cu⁺ ions, theso-called Fenton reaction. ##STR2##

These transition metal ions may also catalyze a reaction betweenhydrogen peroxide and superoxide leading to hydroxyl radical production,the so-called Haber-Weiss reaction. ##STR3##

Ionizing radiation cleaves water with formation of hydrogen atoms andhydroxyl radicals. The hydroxyl radicals so formed account for most ofthe biological damage caused by ionizing radiation.

It appears from the above description that several of the oxygenreduction products are normally formed at the same time. In the xanthineoxidase system, for example, not only is superoxide formed, but alsohydrogen peroxide, both directly and by superoxide dismutation. Thesecompounds may then react further to form the hydroxyl radical. Thexanthine oxidase system can be demonstrated to damage proteins,carbohydrates and nucleic acids and to kill cells. Of the biochemicalcompounds, polyunsaturated lipids appear to be the most sensitive tooxygen toxicity. The oxygen intermediates can initiate chain reactionsinvolving molecular oxygen, the so-called lipid peroxidation. The lipidhydroperoxides so formed and their degradation products not only impairthe function of the cell membranes, but may also damage other cellcomponents.

Organisms living in the presence of oxygen have been forced to develop anumber of protective mechanisms against the toxic oxygen reductionmetabolites. The protective factors include superoxide dismutases (SOD)which dismutate the superoxide radical and are found in relativelyconstant amounts in mammalian cells and tissue. The best known of theseenzymes is CuZn SOD which is a dimer with a molecular weight of 33,000containing two copper and two zinc atoms. CuZn SOD is found in thecytosol and in the intermembrane space of the mitochondria. Mn SOD is atetramer with a molecular weight of 85,000 containing 4 Mn atoms, and ismainly located in the mitochondrial matrix. Until recently, theextracellular fluids were assumed to lack SOD-activity.

However, the present inventors have recently demonstrated the presenceof a superoxide dismutase in extracellular fluids (e.g. blood plasma,lymph, synovial fluid and cerebrospinal fluid) which was termed EC-SOD(extracellular superoxide dismutase). In humans, the activity per ml ofplasma is less than 1% of the total SOD-activity per g of tissue, but itseems to be actively regulated by the body (Marklund et al., Clin. Chim.Acta 126, 1982, pp. 41-51). The affinity for lectins (cf. Example 12)indicates that, contrary to CuZn SOD, the enzyme is a glycoprotein. Itseems to be composed of four equal non-covalently bound subunits with atotal (tetramer) molecular weight of 135,000, and with a metal contentof one Cu atom and one Zn atom per subunit (cf. Example 14). The enzymecatalyzes a first-order dismutation of the superoxide radical, as doother Cu-containing SODS. ##STR4##

The specific activity is very high and is probably mediated by the fourCu atoms of the molecule. Upon chromatography on heparin-Sepharose®, theenzyme is divided into three fractions, A without any affinity, B with aweak affinity and C with a strong heparin affinity. Unlike the behaviourof EC-SOD, CuZn SOD and Mn SOD do not bind to heparin-Sepharose®. Theenzyme has a certain hydrophobic character which may indicate anaffinity for cell membranes. Affinity for heparin often indicatesaffinity for heparan sulfate which is found on cell surfaces, especiallyon vessel endothelium. It may therefore be assumed that EC-SOD is partlylocalized on cell surfaces and partly in extracellular fluids (cf.Example 9-11 below). The amino acid composition and the antigenicreactivity is quite unlike that of the hitherto investigated SODisoenzymes (Marklund, Proc. Nat. Acad. Sci. USA 79, 1982, pp. 7634-7638;Biochem. J. 220, 1984, pp. 269-272). The messenger RNA encoding EC-SODcomprises a sequence coding for a signal peptide (cf. Example 8),indicating that EC-SOD is a secreted protein. The mRNA coding for CuZnSOD, on the other hand, does not comprise such a sequence coding for asignal peptide. Furthermore, the amino acid sequence of EC-SOD isdifferent from that of the other SOD isoenzymes; EC-SOD has been shownto be present in the plasma of all the mammalian species examined(Marklund, J. Clin. Invest. 74, 1984, pp. 1398-1403: Biochem. J. 222,1984, pp. 649-655) as well as in birds and fish. The content varieswidely between the species, but intraspecies variation is very small.Rodent plasma contains 10-20 times more EC-SOD than human plasma whichcontains comparatively little EC-SOD. EC-SOD has also been found in allthe different types of animal tissue examined (Marklund, J. Clin.Invest. 74, 1984, pp. 1398-1403: Biochem. J. 2221 1984, pp. 649-655 ).In tissues the interspecies differences are far smaller. The level ofEC-SOD in tissues (units per gram of wet weight) is higher than thelevel of plasma EC-SOD (units/mi) in humans. In rodents, the tissue andplasma contain approximately equal amounts of EC-SOD (Marklund, Biochem.J. 222, 1984, pp. 649-655).

The activity of the SODs make them interesting candidates fortherapeutic agents to counteract the toxic effects of the superoxide andother oxygen radicals.

Because of the above-mentioned low level of SOD activity inextracellular fluids, the components in the extracellular fluids and thecell surfaces are far less protected against superoxide radicals and theother toxic oxygen reduction products than the cell interior. EC-SODtherefore constitutes a particularly interesting substance fortherapeutic applications in connection with extracellular superoxideradical production.

It would therefore be advantageous to provide EC-SOD in realisticquantities for therapeutic purposes from an easily available source.Such sources which primarily comprise specific types of cells have notpreviously been suggested or described.

Hence, in an important aspect, the present invention relates to EC-SODof recombinant origin. In the present context, the term "recombinant" isintended to indicate that the EC-SOD is derived from a cell which hasbeen subjected to recombinant DNA techniques, i.e. into which a DNAsequence coding for EC-SOD has been inserted and which has subsequentlybeen induced to express EC-SOD. More particularly, the invention relatesto EC-SOD which has a polypeptide structure encoded by the following DNAsequence ##STR5## or any modification thereof encoding a polypeptidewhich has the superoxide dismutating property of native EC-SOD. itshould be noted that the amino acid sequence derived from the DNAsequence is shown above the DNA sequence. Examples of suitablemodifications of the DNA sequence are nucleotide substitutions which donot give rise to another amino acid sequence of the protein, but which,for instance, correspond to the codon usage of the specific organism inwhich the sequence is inserted; nucleotide substitutions which give riseto a different amino acid sequence and therefore, possibly, a differentprotein structure without, however, impairing the critical property ofsuperoxide radical dismutation; a subsequence of the sequence shownabove encoding a polypeptide which has retained the superoxidedismutating property of the native protein; or a DNA sequencehybridizing to at least part of a DNA probe prepared on the basis of thesequence shown above, provided that it encodes a polypeptide which hasthe biological property of superoxide radical dismutation of the nativeprotein. In this connection, it should be noted that the term"superoxide dismutating property of native EC-SOD" and related termsshould be understood to be qualitative rather than quantitative, thatis, relating to the nature rather than the level of activity of thepolypeptide.

The DNA sequence encoding EC-SOD or modifications or derivatives thereofas defined above may be of complementary DNA (cDNA) origin, that is,obtained by preparing a cDNA library on the basis of mRNA from cellsproducing EC-SOD by means of established standard methods end vectors.Hybridization experiments may then be carried out using syntheticoligonucleotides as probes to identify the cDNA sequence coding forEC-SOD. Alternatively, the DNA sequence may be of genomic origin, thatis, derived directly from a cellular genome, for instance by screeningfor genomic sequences hybridizing to a DNA probe prepared on the basisof the full or partial amino acid sequence of EC-SOD found in, forinstance, tissue in accordance with conventional methods; cf. Example 8for a more detailed description of the general procedure. Genomic DNAdiffers from cDNA, for instance by containing transcriptional controlelements and the so-called introns which are non-coding sequences withinthe coding DNA sequence, the significance of which is, at present,obscure. Both cDNA and genomic DNA may be of animal, in particularmammalian, origin. For therapeutic purposes involving human beings, itwill usually be preferred that the EC-SOD is EC-SOD encoded by a DNAsequence of human origin, in order to substantially avoid undesirableadverse immune reactions.

The DNA sequence may also be of synthetic origin, i.e. preparedsynthetically by established standard methods, e.g. as described byMatthes et al., EMBO Journal 3, 1984, pp. 801-805. Finally, the DNAsequence may be of mixed synthetic and genomic origin, mixed genomic andcDNA origin or mixed cDNA and synthetic origin prepared by ligatingtogether DNA fragments of cDNA, genomic or synthetic origin (asappropriate), which DNA fragments comprise part of the gene encodingEC-SOD, in accordance with standard methods.

Although EC-SOD of recombinant origin is preferred in accordance withthe present invention because it is easily available in largequantities, EC-SOD is also available from other sources. Thus, theinvention also relates to EC-SOD of cell line origin, i.e. derived froma cell line producing the protein in significant quantities, such as acell line derived from blood or lung, blood vessel, pancreas, uterus,prostate gland, placenta or umbilical cord tissue or, possibly,neoplastic tissue. Endothelial cells or fibroblasts are at presentcontemplated to be possible sources of EC-SOD.

Finally, the EC-SOD may also be derived from tissue found to berelatively rich in EC-SOD. Accordingly, the present invention furtherrelates to EC-SOD of placenta or umbilical cord origin as these tissueshave been formed to contain reasonably large amounts of EC-SOD comparedto other types of tissue, and are also more easily available than, forinstance, lung, uterus or pancreas tissue. It should be stressed,however, that even though these tissues contain relatively largeramounts of EC-SOD, these are far smaller than those obtainable byrecombinant DNA techniques, and therefore, placenta or umbilical cordEC-SOD is particularly indicated for special purposes requiring onlyminor amounts of EC-SOD.

In a further aspect, the present invention relates to a replicableexpression vector which comprises a DNA sequence encodin EC-SOD. In thepresent context, the term "replicable" means that the vector is able toreplicate in a given type of host cell into which it has beenintroduced. The vector may be one carrying the DNA sequence shown aboveor any suitable modification thereof as explained above. Immediatelyupstream of this sequence (the coding sequence of EC-SOD) there may beprovided a sequence coding for a signal peptide, the presence of whichensures secretion of the EC-SOD expressed by host cells harbouring thevector. The signal sequence may, for instance, be the followingsequence: ##STR6##

It should be noted that this signal sequence (and the signal peptideencoded by it) in itself forms an aspect of the present invention, andit is contemplated that it may be inserted upstream of DNA sequencescoding for other proteins or peptides so as to obtain secretion of theresulting products from the cells.

The vector may be any vector which may conveniently be subjected torecombinant DNA procedures, and the choice of vector will often dependon the host cell into which it is to be introduced. Thus, the vector maybe an autonomously replicating vector, i.e. a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication; examples of such a vector are a plasmid, phage,cosmid, mini-chromosome or virus. Alternatively, the vector may be onewhich, when introduced in a host cell, is integrated in the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

In a further aspect, the invention relates to a cell line which iscapable of secreting EC-SOD. While various human cell lines derived froma wide variety of tissue cell as well as tumour cell lines havepreviously been analysed for their content of EC-SOD (cf. Marklund, J.Clin. Invest. 74, October 1984, pp. 1398-1403), no conclusive resultswere obtained. Although it is mentioned that minor amounts of EC-SOD wasfound in some of the investigated cell lines, the data presented inTable II of the article are not significant and might equally well beascribable to an analytical error. Certainly, a person skilled in theart would not conclude, on the basis of the data presented in thispublication, that some of the cell lines tested for their content ofEC-SOD might possibly produce EC-SOD as a secreted protein, as there isno indication of this possibility in the article.

The cell line may be of mammalian, in particular human, origin. It ispreferable that the cell line is one which produces particularly highquantities (compared to other cells) of EC-SOD. Thus, the cell line maybe one which is derived from blood or lung, skin, blood vessel,pancreas, uterus, prostate gland, placenta or umbilical cord tissue or,possibly, neoplastic tissue. In particular, it is contemplated that celllines derived from fibroblasts or endothelial cells are particularlyadvantageous as sources of EC-SOD, as they are derived from cellsdirectly exposed to extracellular space, which may therefore be assumedto need the protection conferred by EC-SOD from superoxide radicalspresent or generated in an extracellular environment.

The present invention further relates to a cell harbouring a replicableexpression vector as defined above. In principle, this cell may be ofany type of cell, i.e. a procaryotic cell such as a bacterium, aunicellular eukaryotic organism, a fungus or yeast, or a cell derivedfrom a multicellular organism, e.g. an animal or a plant. It is,however, believed that a mammalian cell may be particularly capable ofexpressing EC-SOD which is, after all, a highly complex molecule whichcells of lower organisms might not be able to produce. One example ofsuch a cell is CHO-K1/pPS3neo-18 deposited on Aug. 27, 1986 in theEuropean Collection of Animal Cell Cultures under the Accession NumberECACC 86082701.

The invention also relates to a DNA fragment which encodes EC-SOD andwhich has the following DNA sequence ##STR7##

It should be noted that this sequence includes thesignal-peptide-encoding sequence shown above. The signal sequenceextends from amino acid -18 to -1. The sequence encoding mature EC-SODis initiated at amino acid +1.

The DNA sequence may be of cDNA, genomic or synthetic origin, or ofmixed cDNA and genomic, mixed cDNA and synthetic or mixed cDNA andsynthetic origin as discussed above.

In a further, important aspect, the present invention relates to amethod of producing EC-SOD, in which a cell line producing EC-SOD isgrown under conditions ensuring the secretion of EC-SOD. The cell linemay be derived from any of the sources mentioned above. Mammalian cellsproducing EC-SOD may be identified immunohistochemically by means ofantibodies directed against EC-SOD or by analysing for EC-SOD secretedinto the medium in which specific cells are cultured. The cells may begrown in conventional media adapted to the propagation of cell lines ina manner known per se.

As mentioned above, EC-SOD shows an affinity to heparin which indicatesan affinity to heparan sulphate or other heparin-likeglucoseaminoglucanes found on cell surfaces, especially on the surfaceof endothelial cells. It is therefore contemplated to induce the releaseof EC-SOD from cell surfaces and thereby ensure an improved yield ofEC-SOD by growing the tissue cells of the EC-SOD producing cell lines ina medium containing heparin or a heparin analogue, e.g. heparansulphate, or another sulphated glucoseaminoglycane, dextran sulphate oranother strongly negatively charged compound in an amount which issufficient to induce the release of EC-SOD from the cell surfaces (cf.Example 9-11).

In another important aspect, the invention relates to a method ofproducing EC-SOD, in which a DNA fragment comprising a DNA sequenceencoding EC-SOD is inserted in a vector which is able to replicate in aspecific host cell, the resulting recombinant vector is introduced intoa host cell which is grown in or on an appropriate culture medium underappropriate conditions for expression of EC-SOD, and the EC-SOD isrecovered. The medium used to grow the cells may be any conventionalmedium suitable for the purpose, but it may be necessary to add extra Cuand/or Zn for the synthesis of EC-SOD, especially if it is to beproduced in increased amounts. A suitable vector may be any of thevectors described above, and an appropriate host cell may be any of thecell types listed above. The methods employed to construct the vectorand effect introduction thereof into the host cell may be any methodsknown for such purposes within the field of recombinant DNA. The EC-SODexpressed by the cells may be secreted, i.e. exported through the bellmembrane, dependent on the type of cell and the composition of thevector. If the EC-SOD is produced intracellularly by the recombinanthost, that is, is not secreted by the cell, it may be recovered bystandard procedures comprising cell disrupture by mechanical means, e.g.sonication or homogenization, or by enzymatic or chemical means followedby purification (examples of the recovery procedure are given inExamples 1, 4-6 and 17).

In order to be secreted, the DNA sequence encoding EC-SOD should bepreceded by a sequence coding for a signal peptide, the presence ofwhich ensures secretion of EC-SOD from the cells so that at least asignificant proportion of the EC-SOD expressed is secreted into theculture medium and recovered. It has been experimentally establishedthat part of the secreted EC-SOD is present in the medium and part ofthe EC-SOD is present on the cell surfaces. Hence, the expression"secreted into the culture medium" is intended to encompass anytransport of the EC-SOD through the cell membrane, whether the enzymeeventually ends up in the culture medium or on the cell surfaces (cf.Examples 9-11). The EC-SOD may be recovered from the medium by standardprocedures comprising filtering off the cells and isolating the secretedprotein, for instance as outlined in Examples 1, 4-6 and 17. In order toensure the release of EC-SOD from the cell surfaces, and thus obtain animproved yield, it may be advantageous to add heparin or one of thesubstances with a similar effect mentioned above to the medium asexplained above.

In a still further aspect, the present invention relates to a method ofrecovering EC-SOD, in which an extract of a biological materialcontaining EC-SOD activity is adsorbed to a matrix containingimmobilized antibodies against EC-SOD or an immunological determinantthereof, the EC-SOD activity is eluted from the matrix, and thefractions containing the EC-SOD activity are pooled, optionally followedby further purification. The antibodies employed may be antibodiesraised against EC-SOD or an immunological determinant thereof andimmobilized on a suitable matrix material in a manner known per se.

The antibodies employed for affinity chromatography according to theinvention may either be polyclonal or monoclonal antibodies. Thecurrently preferred antibodies are monoclonal antibodies as mostmonoclonal antibodies have been found to bind the antigen less stronglythan the polyclonal antibody mixture which means that desorption may becarried out under milder conditions with weaker eluants. This may resultin an improved yield of EC-SOD as there is a lower degree ofdenaturation than when strong eluants are used for desorption.

Also, since all the IgG will be directed against EC-SOD, a far smalleramount of antibody matrix will have to be used for the adsorption ofEC-SOD from the biological material used as the starting material. Thedesorption of EC-SOD will require a far smaller volume of eluant,thereby simplifying the elution procedures which are at presentinconvenient due to the large volumes of eluant needed for thedesorption.

The specificity of monoclonal antibodies for EC-SOD is likely to behigher than that of the polyclonal antibodies. The eluate from theantibody matrix will therefore be purer which means that one or morefurther purification steps may be omitted. This means that theproduction procedure will be greatly simplified and a far higher yieldof EC-SOD obtained from the same quantity of starting material whichpresents an important economic advantage.

Polyclonal antibodies may be obtained by immunizing an immunizableanimal with EC-SOD or an immunological determinant thereof and obtainingantiserum such as immunoglobulins from the animal in a manner known perse. The immunization is preferably be performed by means of a stabilizedaqueous solution of EC-SOD; the stabilization agent may be a buffer suchas phosphate buffered saline or an adjuvant (also to further increasethe antigenicity), and a suitable adjuvant is Freund's adjuvant oraluminium hydroxide. For immunization purposes, mice, rabbits, hens,goats and sheep are the preferred animals. The bleeding of the animaland the isolation of the antiserum is performed according to well-knownmethods. The anti body is preferably provided in substantially pure formwhich is advantageous in order to obtain the necessary purification ofthe EC-SOD.

When using a monoclonal antibody in the method of the invention, it maybe produced by a hybridoma technique which is well-known method forproducing antibodies. In the hybridoma technique using, for instance,mice as the animals immunized, mice are immunized with the antigen inquestion and spleen cells from the immunized mice are fused with myelomacells whereupon the fused hybridoma cells are cloned, antibody-producingcells are grown in a suitable growth medium and the antibodies arerecovered from the culture. The antibodies obtained by the hybridomatechnique have the advantage of greater specificity and, hence, agreater purifying potential as mentioned above. In a possible furtherstep, using recombinant DNA techniques, the DNA sequence encoding theantibody is transferred from the hybridoma cell clone to a suitablevector, the hybrid vector is transformed to a suitable bacterial host,the host is grown in an appropriate medium and the resulting antibody isrecovered from the culture. In this way, an improved yield of antibodymay be obtained. The host may be one usually employed in the field ofrecombinant DNA technology such as Eschericia coli or Bacillus subtilis.

In an alternative method of obtaining monoclonal antibodies, thehybridoma cells may be implanted and grown in the abdominal cavity ofmice, and the resulting anti-EC-SOD antibodies are recovered from theascitic fluid produced, in a manner known per se. Furthermore,immunization for obtaining monoclonal antibodies may be performed invitro using immunocompetent cells from, e.g., mice. In this case it isnot necessary to inject an antigen into the animal in question, e.g. amouse, although this is at present the most commonly employed procedure.It should be noted that monoclonal antibodies and the method ofpreparing them form one aspect of the invention.

The biological material from which the extract containing the EC-SODactivity is obtained may, for instance, be mammalian tissue. It may bepossible to employ bovine, porcine or equine tissue as, for the purposeof comparison, bovine CuZn SOD has been found to have relatively littleimmunological reactivity in humans and allergic reactions have not beenany serious problem. On the basis of this, it may therefore be concludedthat the same will be the case for bovine, equine or porcine EC-SOD. Itis, however, still preferred to employ human tissue in order to obviatepossible undesirable immunological reactions. Human tissue currentlypreferred for this purpose as it contains relatively large amounts ofEC-SOD is human lung, pancreatic, uterine, prostate, umbilical cord orplacental tissue, especially the latter two types of tissue, as theseare also more easily available. The extract may be prepared in aconventional manner in a suitable buffer such as a phosphate buffer. Ithas been found advantageous to add a chaotropic salt, e.g. KBr, ammoniumsulphate or potassium thiocyanate, to the buffer in order to improve theyield of EC-SOD from the extraction.

However, since EC-SOD is not produced in large quantities in any ofthose tissues so that very high quantities of tissue will be needed toproduce a sufficient amount of EC-SOD for therapeutic purposes, it maybe preferred to obtain EC-SOD from an animal, preferably a mammalian,human, cell line which produces EC-SOD. The biological materialcontaining EC-SOD activity may therefore also be the material resultingfrom growing a cell line producing EC-SOD, as described above. In orderto obtain particularly high amounts of EC-SOD, the EC-SOD to berecovered by the methods of the invention may advantageously be producedby recombinant DNA methods as described above.

In most case, especially when employing immobilized polyclonalantibodies for the purification of EC-SOD, the pooled eluate of theantibody matrix may be absorbed to a matrix, e.g. an ion-exchangematrix, followed by eluting the EC-SOD activity from the matrix andpooling fractions containing the EC-SOD activity. Further purificationof the pooled eluate may be obtained by applying it on a chromatographiccolumn of a matrix comprising heparin or a heparin analogue, e.g.heparan sulphate, or another sulphated glucoseaminoglycane, dextransulphate or another strongly negatively charged compound and elutingfollowed by pooling the fractions showing affinity to the substance inquestion.

In a yet further, very important aspect, the present invention relatesto the use of EC-SOD for the diagnosis, prophylaxis or treatment ofdiseases or disorders connected with the presence or formation ofsuperoxide radicals and other toxic oxygen intermediates derived fromthe superoxide radical.

Examples of such diseases or disorders are selected from conditionsinvolving ischaemia followed by reperfusion, e.g. infarctions such asheart, kidney, brain or intestine infarctions, inflammatory diseasessuch as rheumatoid arthritis, pancreatitis, in particular acutepancreatitis, pyelonephritis and other types of nephritis, andhepatitis, autoimmune diseases, insulin-dependent diabetes mellitus,disseminated intravascular coagulation, fatty embolism, adultrespiratory distress, infantile respiratory distress, brain haemorrhagesin neonates, burns, adverse effects of ionizing radiation, andcarcinogenesis.

Thus, EC-SOD is indicated for substantially the same applications asCuZn SOD the therapeutic activity of which has been more thoroughlydocumented as discussed below.

EC-SOD, however, has been found to possess a number of characteristicswhich are assumed to make it particularly useful for therapeuticapplications. CuZn SOD has a low molecular weight (33,000) which causesit to become eliminated very quickly by glomerulus filtration in thekidneys so that, in human beings, the enzyme has a half-life of onlyabout 20-30 minutes. Preliminary experiments with EC-SOD havesurprisingly established a significantly longer half-life of EC-SOD (inrabbits), cf. Example 10. This is currently believed partly to beascribable to the high molecular weight of EC-SOD, 135,000, whichprevents it from being eliminated by glomerulus filtration, and partlyto the fact that EC-SOD seems to bind to endothelial cell surfaces asindicated below. In the therapeutic use of EC-SOD according to theinvention, the enzyme therefore preferably has a half-life in humanbeings of at least 4 hours and possibly even longer.

As explained above, EC-SOD is, in its native environment, a secretedprotein and it is therefore likely that it is specifically synthesizedfor a function in extracellular space (in extracellular fluids or oncell surfaces) which may cause it to exhibit properties which areparticularly well adapted to protect plasma components or the outersurface of cells against the toxic effects of superoxide radicals orother oxygen radicals. This suggestion is supported by the findings thatEC-SOD has a slightly hydrophobic character which may cause it to have atendency to bind to the outer surface of cells, and that it showsaffinity to heparin indicating an affinity to heparan sulfate which isfound on the outer surface of cells, both of which qualities would seemto indicate a particularly good ability to protect tissue, cf. Example9, 10 and 11 in which the test results seem to verify the binding ofEC-SOD to blood vessel endothelium.

The significance of the apparent association of EC-SOD with cellmembranes is further supported by the finding that CuZn SOD which hasbeen modified with polylysine to bind to cell membranes is better ableto protect activated (superoxide radical-producing) polymorphonuclearleukocytes (PMN) from autoinactivation (cell death) than normal CuZn SODwhich is negatively charged and therefore tends to be repelled by thecell membranes (M. Salin and J. M. McCord, "Free radicals in leukocytemetabolism and inflammation", in Superoxide and Superoxide Dismutases,eds. A. M. Michelson, J. M. McCord and I. Fridovich, Academic Press,1977, pp. 257-270). The fact that Nocardia asteroides possesses amembrane-associated SOD which appears to confer efficient protectionagainst activated human PMNs as the susceptibility of Nocardia to PMNsis significantly increased when Nocardia cells are treated withantibodies towards this SOD (B. L. Beaman et al., Infection and Immunity47, 1985, pp. 135-141) also points to a cell membrane-protectivefunction of SOD bound to cell surfaces. Unlike EC-SOD, CuZn SOD has anintracellular function which may make it less well suited forextracellular application, i.e. occasioned by the extracellular presenceof superoxide radicals. Furthermore, its brief half-life compared tothat of EC-SOD mentioned above would seem to make it necessary toadminister larger doses at shorter intervals that is likely to be thecase with EC-SOD.

SOD activity for potential therapeutic applications has beendemonstrated for the following diseases or disorders.

Parenterally administered CuZn SOD has been shown to exhibit ananti-inflammatory effect in a series of animal models of inflammation aswell as in inflammatory diseases in animals (Huber and Saifer, inSuperoxide and Superoxide Dismutoses, eds. Michelson et al., AcademicPress, 1977, pp. 517-549). In humans, positive effects of CuZn SOD hasbeen reported in rheumatoid arthritis and arthroses, in inflammation ofthe bladder and other urological conditions (Menander-Huber inBiological and Clinical Aspects of Superoxide and Superoxide Dismutase,eds. Bannister et al. , Elsevier/North Holland, 1980, pp. 408-423) aswell as adverse effects of treatment with ionizing radiation (Edsmyr etal., Current Therapy. Res. 10, 1976, pp. 198-211; Cividalli et al.,Acta. Radiol. Oncol. 24, 1985, pp. 273-277 (in rats)). In somecountries, bovine CuZn SOD has become registered as a drug (Orgotein,Peroxinorm), employed mainly for the treatment of arthritis andarthroses where the composition is administered intraarticularly (Goebeland Storck, Am. J. Med. 74, 1983, pp. 124-128).

Parenterally administered CuZn SOD is not taken up by the cells and mustexert its activity in extracellular space. CuZn SOD encapsulated inliposomes is taken up by the cells and is reported to be effectiveagainst Crohn's disease, Bechet's disease, dermatitis herpetiformis,ulcerative colitis, Kawasaki's disease and against the adverse effectsof radiation therapy (Y. Niwa et al., Free Rad. Res. Comms. 1, 1985, pp.137-153). The mechanism of the anti-inflammatory activity of CuZn SOD isnot quite clear. Direct protection against oxygen radicals formed byactivated leucocytes has been suggested (Halliwell, Cell Biol. Int. Rep.6, 1982, pp. 529-541). Another possibility is prevention of theformation of a superoxide induced strongly chemotaxic substance (Petroneet al., Proc. Natl. Acad. Sci. USA 77, 1980, pp. 1159-1163).

The other large potential area of application for SOD is as a protectivefactor against tissue damage caused by ischaemia followed byreperfusion. If the supply of blood to a tissue is cut off, the tissuewill slowly become necrotic. Macro- and microscopically the damage willtypically develop slowly over several hours. If the tissue is reperfusedafter, for instance, one hour, a strong acceleration of the tissuedamage will occur instead of an improvement. Most likely there areseveral reasons for this so-called reperfusion paradox, but oxygenradicals formed as a result of the reappearance of oxygen in previouslyischaemic tissue appear to contribute to the damage. Since the radicalsare extremely shortlived and therefore difficult to study directly,their formation and effects may be inferred from the protective actionof various scavengers. Tissue protection (by means of a protectivesubstance) has been demonstrated in ischaemia- or anoxiareperfusionmodels in the kidney (SOD [G. L. Baker et al., Am. Surg. 202, 1985, pp.628-641; I. Koyama et al., Transplantation 40, 1985, pp. 590-595], SOD,catalase [E. Hansson et al., Clin. Sci. 65, 1983, pp. 605-6101] andallopurinol .[G. L. Baker et al., op. cit.; I. Koyama et al., op. cit.])intestine (SOD [D. A. Parks et al., Gastroenterology 82, 1982, pp. 9-15;M. H. Schoenberg et al., Acta Chim. Scand. 150, 1984, pp. 301-309; M. C.Daising et al., J. Surg. Res. 34, 1983, pp. 589-596] and allopurinol [D.A. Parks et al., op. cit.]), pancreas (SOD, SOD+catalase, catalase andallopurinol [H. Sanfey et al., Ann. Surg. 200, 1983, pp. 405-413]),liver (SOD and catalase [S. L. Atalla et al., Transplantation 40, 1985,pp. 584-589], lung (SOD+catalase [R. S. Stuart et al., Transplant. Proc.17, 1985, pp. 1454-1456]) skelbtal muscle (SOD, catalase and allopurinol[R. V. Korthuis, Circ. Res. 57, 1985, pp. 599-609]), skin (SOD andallopurinol [M. J. Im et al., Ann. Surg. 201, 1985, pp. 357-359]) andbrain (SOD and allopurinol [J. S. Beckmann et al., in Superoxide andSuperoxide Dismutase in Chemistry, Biology and Medicine, ed. G. Rotilio,Elsevier, 1986, pp. 602-607]).

Preservation of heart function has been reported in isolated, perfusedpreparations from the rabbit (catalase, allopurinol; SOD had no effect[C. L. Myers et al., J. Thorac. Cardiovasc. Surg. 91, 1986, pp.281-2891]), cat (SOD+catalase [M. Schlafer et al., Circulation 66,Suppl., 1982, pp. 185-192]) and rat (glutathione, catalase, SOD Gaudelet al., J. Mol. Cell Cardiol. 16, 1984, pp. 459-470]). In regionalischaemia-reperfusion models in vivo, reduction of infarct size has beenreported in the dog (SOD [T. J. Gardner et al., Surgery 94, 1983, pp.423-427; D. E. Chambers et al., J. Mol. Cell Cardiol. 17, 1985, pp.145-152; S. W. Werns et al., Circ. Res. 56, 1985, pp. 895-898]), andallopurinol [D. E. Chambers et al., op. cit.; T. J. Gardner et al., op.cit.] catalase had no effect [S. E. Werns et al., op. cit.] Injection ofSOD+catalase has also been reported to preserve the mechanical heartfunction after a brief (15 minutes) regional ischaemia in the dog (M. L.Myers et al., Circulation 72, 1985, pp. 915-921; K. Przyklenk and R. A.Kloner, Circ. Res. 58, 1986, pp. 148-156). Furthermore, SOD has beenreported to reduce the incidence of ischaemia-and-reperfusion inducedarrythmias (B. Woodward et al., J. Mol. Cell. Cariol. 17, 1985, pp.485-493; M. Bernier et al., Circ. Res. 58, 1986, pp. 331-340). Thesource of oxygen radicals in this situation is not completely clear, butthe effect of allopurinol seems to indicate that it is partly caused byxanthine oxidase which, by ischaemia, is converted from its xanthindehydrogenase form (Parks et al., op. cit.) to the radical-producingoxidase form. A large amount of hypoxanthine which is the substrate forxanthine oxidase is formed due to purine nucleotide degradation inducedby ischaemia. Other sources of superoxide radicals may be activatedleukocytes attracted to ischaemia-damaged tissue, prostagiandinsynthesis (O₂.⁻ is a byproduct; Kontos, Circ. Res. 57, 1985, pp.508-516) and autooxidation of various compounds accumulated in reducedform during ischaemia. The finding concerning ischaemia followed byreperfusion has potentially important clinical applications. It may bepossible to obtain an excellent effect by reperfusion of tissue inconnection with heart infarctions, by the concomitant administration ofan SOD and/or other protective factors against oxygen radicals andthrombolytic factors, e.g. tissue plasminogen activator. The results ofthe SOD experiments also indicate a possible application in connectionwith heart surgery and heart transplantations. Analogously, the resultsof employing an SOD in connection with kidney ischaemia followed byreperfusion may be employed in connection with kidney transplantationsand other organs transplantations such as skin, lung, liver, or pancreastransplantations. Ischaemia brain disease is another possibleindication.

SODs show interesting protective effects in connection with otherpathological conditions.

Thus, pancreatitis was induced in dog pancreas in three different ways:infusion of oleic acid, partial obstruction of the excretory ducts andischaemia followed by reperfusion. SOD, catalase, and SOD+catalase werefound to be protective, the combination treatment being generally themost effective. In the ischaemia model, however, SOD alone was almost aseffective as the combination of SOD and catalase (Sanfey et al., Ann.Surg. 200, 1984, pp. 405-413). SOD+catalase has been reported toameliorate pancreatitis induced by cerulein in rats (K. S. Guice et al.,Ann. J. of Surg. 151, 1986, pp. 163-169). The results indicate thepossibility of an active therapy against this disease for which nospecific therapy exists at present.

It has also been suggested that treatment with SOD is effective againstburns. The local oedema after an experimental slight burn in rats couldbe somewhat decreased through injection of SOD (Bjork and Artursson,Burns 9, 1983, pp. 249-256). In an animal model involving severe burndamage of mice a dramatic protection was obtained by means of SOD, wheresurvival and local damage were concerned (Saez et al., Circulatory Shock12, 1984, pp. 220-239).

In the case of for instance, burns, immunocomplex formation, and majortissue damage, neutrophilic leukocytes are accumulated in the lungs.Complement activation (C5 a) often seems to mediate the accumulation.The leukocytes seem to be activated and release oxygen radicals, therebycausing lung damage which, for instance, is characterized by increasedvessel permeability and lung oedema (adult respiratory distress). Inseveral animal models, SOD and other oxygen radical scavengers have beenshown to have a protective effect against lung damage (Till and Ward,Agents and Actions, Suppl. 12, 1983, pp. 383-396).

Parenterally administered CuZn SOD has been reported to preventbronchopulmonary dysplasia in preterm neonates suffering from infantilerespiratory distress (W. Rosenfeld et al., J. Pediatr. 105, 1984, pp.781-785).

In a beagle puppy model, injection of SOD has been reported to reducethe frequency of intraventricular brain haemorrhage followinghypotension (L. R. Ment et al., J. Neurosurg. 62, 1985, J63-J69).

SOD ameliorates hepatitis in rats induced by injection ofCorynebacterium parvum (M. J. P. Arthur et al., Gastroenterology 89,1985, pp. 1114-1122).

The endothelium-derived vessel relaxant factor is very sensitive tosuperoxide, and administration of SOD augments its actions (R. J.Gryglewski et al., Nature 320, 1986, pp. 454-456; G. M. Rubanyi et al.,Am. J. Physiol. 250, 1986, pp. H822-H827). Superoxide radical productioncan occur under many circumstances in the body and may causevasoconstriction and decreased tissue perfusion. Administration of SODis believed to be able to relieve such vasoconstriction.

Acute severe increase in blood pressure leads to functional andmorphologic absormalities in brain arterioles. Prostaglandin synthesisinhibitors and superoxide dismutase is contemplated to protect againstthe abnormalities. Superoxide release can be detected (H. A. Kontos,Circ. Res. 57, 1985, pp. 508-516). Close analysis of the model has leadto the conclusion that superoxide radicals are formed as a by-productduring prostaglandin synthesis. The results suggest that tissue damagecaused by superoxide radicals released during prostaglandin synthesismay occur in other pathological situation and that SOD may exert aprotective action.

CuZn SOD+catalase in the medium have been reported to prolong thesurvival of the perfused isolated rabbit cornea (O. N. Lux et al., Curr.Eye Res. 4, 1985, pp. 153-154). CuZn SOD+catalase protect the isolatedless against photoperoxidation (S. D. Varma et al., Ophthalmic Res. 14,1982, pp. 167-175). The results suggest possible beneficial effects ofSOD in cornea transplantations and other opthalmic surgical procedures.

Ameliorative action of parenteral SOD has been reported in animal modelsof such acute conditions as disseminated intravascular coagulation (T.Yoshikawa et al., Thromb. Haemostas. 50, 1983, pp. 869-872) andsepticemia (H. F. Welter et al., Chirurgisches Forum '85 f. experim. u.klinischer Forschung, Springer-Verlag, Berlin, 1985).

In various types of autoimmune disease, such as systemic lupuserythematosus (SLE), systemic sclerosis and rheumatoid arthritis, anincreased frequency of chromosomal breaks in lymphocytes has beendemonstrated (Emerit, "Properties and action mechanisms of clastogenicfactors", in Lymphokines, Vol. 8, ed. E. Pick, Academic Press, 1983, pp.413-424). Fibroblast cultures and direct bone marrow preparations alsosometimes show increased breakage. Plasma from such patients contains achromosome breaking--clastogenic--factor. In some instances a similarfactor has also been demonstrated in synovial fluid and in cerebrospinalfluid (disseminated sclerosis). Breaks occur in normal lymphocytes whichare cocultivated with lymphocytes from patients with autoimmune disease.Lymphocytes from patients condition culture media to produce chromosomebreaks. The clastogenic activity of SLE plasma can be increased byUV-irradiation. Production of superoxide in plasma by means of xanthineoxidase and xanthine results in formation of clastogenic activity. Inall the above described models, addition of CuZn SOD to the mediumprotected against the clastogenic activity (Emerit, ibid.). Thisindicates that superoxide radicals are involved in both the formationand actions of the clastogenic factor (Emerit, Ibid.).

In an animal model of SLE, the New Zealand black mouse which possessesthe clastogenic factor, the chromosomes are protected in bone marrowcells in vivo by repeated injections of SOD (Emerit et al., Cytogenet.Cell Genet. 30, 1982, pp. 65-69). It is, however, still unclear to whatextent the clastogenic factor contributes to the major symptoms in humanautoimmune disease and whether administration of SOD has any therapeuticeffect.

The neoplastic transformation of cells is usually divided into twophases, i.e. initiation followed by promotion. In in vitro models whereinitiation has been caused by ionizing radiation, bleomycin,misonidazole and other nitroimidazoles the oncogenic transformation hasbeen effectively inhibited by the presence of SOD in the medium. It isnot necessary for SOD to be present during exposure to the initiatingsubstances which seems to indicate that the enzyme inhibits thesubsequent promotion step (Miller et al., Int. J. Radiat. Oncol. Biol.Phys. 8, 1982, pp. 771-775; Borek and Troll , Proc. Nat. Acad. Sci. USA80, 1983, pp. 1304-1307). Non-toxic doses of xanthine+xanthine oxidasecauses promotion in growing cells. Addition of SOD or SOD+catalaseinhibits this effect (Zimmerman and Cerutti, Proc. Nat. Acad. Sci. USA81, 1984, pp. 2085-2087). Phorbol esters are known promoters. In a modelin which skin tumors were induced by initiation with a benzanthracenefollowed by application of a phorbol ester (TPA), local treatment with alipophilic copper complex with SOD activity strongly reduced tumorformation (Kenzler et al., Science 221, 1983, pp. 75-77). The resultindicates that, at least in certain cases, superoxide radicalscontribute to the promotion of tumor formation and that SOD may protectagainst this effect.

There is reason to believe that oxygen radicals contribute to thedamaging effects of a number of toxic substances such as bleomycin,adriamycin, alloxan, 6-hydrodopamine, paraquat, dihydroxyfumaric acid,nitrofurantoin and streptozotocin. In those cases where radicalformation takes place in the extracellular space it might be possible toprotect by means of injected protective enzyme. Thus, SOD may protectagainst the diabetogenic activity of alloxan (damages β-cells in thepancreas) in vitro (Grankvist et al., Biochem. J. 182, 1979, pp. 17-25)and in vivo (Grankvist et al., Nature 294, 1981, pp. 158-160). Thedamaging effect of alloxan seems therefore to be mediated by thesuperoxide radical or by other oxygen radicals derived from it. Thereason for the great sensitivity of the β-cells to alloxan is not clear,and it has been speculated whether there is any connection betweenalloxane sensitivity and the incidence of insulin-dependent diabetesmellitus. In diabetes mellitus there is an infiltration in theLangerhans' islets by inflammatory cells which potentially may formoxygen radicals. It may therefore be contemplated to protect the β-cellsby injections with SOD at the first onset of diabetes mellitus.

It has been reported (Mossman and Landesman, Chest 835, 1983, pp.50s-51s) that SOD added to the growth medium protects tracheal cellsagainst asbestos.

It has been described (Roberts et al., J. Urol. 128, 1982, pp.1394-1400) that parenteral CuZn SOD protects kidneys againstexperimentally induced pyeonefritis. SOD and, in particular, catalaseprotect against acute nephrotoxic nephritis induced in rats byantiglomerular basement membrane antibodies (A. Rehan et al., Lab.Invest. 51, 1984, pp. 396-403).

Generally, CuZn SOD has been employed as the test substance in theexperiments described above. It is, however, assumed that EC-SOD may beemployed for the same purposes and, as has been indicated above, withgreater efficiency due to its particular properties which may make itespecially attractive to employ EC-SOD extracellularly.

The present invention further relates to a pharmaceutical compositionwhich comprises EC-SOD together with a pharmaceutically acceptableexcipient, diluent or vehicle. The EC-SOD incorporated in thecomposition may be from any of the sources discussed above, i.e. ofrecombinant, cell line or tissue origin.

The estimate of a suitable, i.e. therapeutically active, dosage forsystemic treatment is made on the basis of the content of EC-SOD in thehuman body. EC-SOD is the major SOD in human plasma, and the totalactivity (composed of fractions A, B, and C, cf. Example 2) is about 20U/ml. Injection of 200 IU heparin per kg body weight results in anincrease of EC-SOD fraction C of about 23 U/ml, cf. Example 9. Althoughthis heparin dosage is very high, a maximum release was apparently notachieved, cf. Example 9. Assuming that approximately twice as muchEC-SOD fraction C may be released from vessel endothelium, the totalEC-SOD content in vessels would correspond to about 66 U/ml plasma(20+2×23 U/ml). The total plasma volume is about 4.7% of the body weightcorresponding to about 3.3 l in a 70 kg person. 1 unit EC-SOD equalsabout 8.8 ng. The total amount of EC-SOD in the blood vessels (plasmaand vessel endothelium) is therefore 3300×66×8.8×10.sup. -9 g=1.92 mg. Atenfold increase would require 19 mg and a 300-fold increase 575 mgEC-SOD C. A suitable dosage of EC-SOD may therefore be in the range ofabout 15-600 mg/day, dependent, i.a. on the type and severity of thecondition for which administration of EC-SOD is indicated. Injection of,for instance, 87 mg EC-SOD C (a 50-fold increase) would result in 26μg/ml in plasma (disregarding endothelium binding). This or even lowerconcentrations show strong protective properties in in vitro experiments(with CuZn SOD) (cf. A. Baret, I. Emerit, Mutation Res. 121, 1983, pp.293-297; K. Grankvist, S. Marklund, J. O. Sehlin, I. B. Taljedal,Biochem. J. 182, 1979, pp. 17-25).

The dosage and timing of EC-SOD injections depends on the half-life ofthe enzyme in human blood vessels, which is not yet known. In rabbitshuman EC-SOD displayed a half-life of about 18 h (cf. Example 10). Thehalf-life in humans is probably longer. Assuming first-order kineticsand a half-life of 36 h, daily injections of 35 mg after an initialinjection of 87 mg would therefore result in the same concentration asafter the initial injection.

Example 9 shows that EC-SOD C can be mobilized from cell surfaces toplasma with heparin. Parenteral heparin, other sulphatedglucoseaminoglycans and other strongly negatively charged substances maybe used to modulate the location of endogenous or injected EC-SOD C (cf.Example 9-11). Localization to the plasma phase might be useful incertain pathological conditions.

EC-SOD is composed of three fractions, A without, B with weak and C withstrong heparin-affinity. The reasons for the different affinities arenot known yet, but the fractions are in most respects very similar.Electrophoresis in PAGE-SDS gels reveals no differences, the amino acidcompositions appear to be identical (S. Marklund, Proc. Natl. Acad. Sci.USA 79, 1982, pp. 7634-7636) and antibodies raised against A and C seemto react equally With all three fractions (S. Marklund, Biochem. J. 220,1984, pp. 269-272). The fractions may be produced by the same gene, andbe modified posttranslationally. All three fractions exist in freshplasma (cf. Example 9) and is the fraction, the pharmacokinetics ofwhich is discussed above. C is also the fraction produced by recombinantDNA techniques (cf. Example 13). However, it is contemplated that EC-SODfractions A and B may also be therapeutically useful in pathologicalconditions in which high SOD activity in solution is important.

For topical treatment, far less EC-SOD than indicated above willprobably be needed. At present, 4-8 mg of CuZn SOD are administeredintraarticularly once a week for the treatment of arthritis. EC-SODwhich has a far higher molecular weight is likely to remain in the jointfor a longer period of time. A similar treatment protocol or possiblysomewhat lower doses will probably be appropriate.

Before use, the EC-SOD-containing composition should preferably bedry-stored, e.g. in lyophilized form. For systemic treatment and localinjections the EC-SOD may suitably be formulated for parenteraladministration, e.g. dissolved in an appropriate, non-toxic,physiologically acceptable solvent such as isotonic saline. For topicalapplication, the pharmaceutical composition may be in the form of, forinstance, an ointment, lotion, spray, cream or aerosol containingEC-SOD.

It is further contemplated that, in the pharmaceutical composition ofthe invention, EC-SOD may advantageously be combined with catalase whichdismutates hydrogen peroxide in the following way: ##STR8##

The combined action of EC-SOD and catalase further reduces the formationof the hydroxyl radical which,, as described above, is thought to be themost toxic of the oxygen radicals. Instead of catalase, anotherantioxidant which cooperates with EC-SOD to reduce the toxic effects ofoxygen reduction products may be employed.

It is also contemplated that combinations of EC-SOD and other substancessuch as allopurinol (inhibits xanthine oxidase), scavengers of thehydroxyl radical (e.g. mannitol or compounds containing the sulfhydrylgroup) and chelators of transition metal ions (e.g. desferrioxamine) maybe advantageous.

Moreover, for applications where the presence of EC-SOD in plasma isindicated, it may be advantageous to incorporate heparin or a heparinanalogue, e.g. heparan sulphate or another sulphatedglucoseaminoglycane, dextran sulphate or another strongly negativelycharged compound, in the composition in order to mobilize the EC-SODpresent on cell surfaces in the patient to be treated, thereby providingan extra dosage of EC-SOD in the plasma.

The invention also relates to a method of preventing or treating adisease or disorder connected with the presence or formation ofsuperoxide radicals, comprising administering, to a patient in need ofsuch treatment, a therapeutically or prophylactically effective amountof EC-SOD. The disease or disorder may be any one of those discussedabove. The invention also relates to a method of preventing or treatingdamage caused by ischemia followed by reperfusion in connection with thetransplantation of organs such as kindney, lung, pancreas, heart, liveror skin, or in connection with heart surgery, comprising administering atherapeutically or prophylactically effective amount of EC-SOD before,during or after surgery. In either method, a therapeutically orprophylactically active dosage may comprise about 15-600 mg/day ofEC-SOD, dependent i.a., on the type and severity of the condition to betreated. It may be advantageous, in this method, to co-administerheparin or another strongly negatively charged compound as discussedabove, and/or catalase or an antioxidant with a similar effect as alsodiscussed above.

DESCRIPTION OF THE DRAWINGS

The invention is further described with reference to the drawings, inwhich

FIG. 1 is a graph showing the elution pattern of protein and EC-SODafter immunoadsorption of an EC-SOD-containing material toanti-EC-SOD-Sepharose® (cf. Example 4).

FIG. 2 is a graph showing the elution pattern of EC-SOD fromDEAE-Sephacel® (cf. Example 5).

FIG. 3 is a graph showing the elution pattern of EC-SOD fromheparin-Sepharose® (cf. Example 6).

FIG. 4 is an autoradiogram showing the hybridization pattern with the48-meric probe of one of the nitrocellulose filters onto which about20,000 λgtll plaques had been transferred. The dark spots on theautoradiogram represent human EC-SODcDNA-containing plaques (cf. Example8).

FIGS. 5a and 5b show the DNA sequence and deduced amino acid sequence ofEC-SOD (cf. Example 8).

FIG. 6 shows the structure of plasmid pPS3. The white areas representSV40 DNA and the numbers refer to the corresponding nucleotide positionsin SV40. SV40PE and SV40 origin represent the SV40 early promotor andthe SV40 origin of replication, respectively, and arrows show thedirection of transcription and DNA replication, respectively. SV40polyadenylation signals are located between positions 2770 and 2533. Thehatched area represents human EC-SOD cDNA. The solid black arearepresents the β-lactamase gene (AP®) of plasmid pBR322. Thin linesrepresent plasmid pBR322 DNA. Also indicated is the location of thepBR322 origin of replication.

FIG. 7 shows the effect of intravenous heparin injection on plasmaEC-SOD. 200 IU heparin per kg body weight was injected at time zero intotwo healthy males and plasma collected before and at indicated timesafter. The EC-SOD activity was determined as described in Example 9.

FIG. 8 shows the dose response of the EC-SOD releasing activity ofintravenous heparin. Heparin was injected intravenously at the indicateddoses into two healthy males and blood was collected before and at 10and 15 minutes after the heparin injection. At 15 minutes an additionaldose of heparin up to 200 IU/kg body weight, was injected and bloodcollected 10 minutes thereafter. EC-SOD was determined in plasma asdescribed in Example 9. The difference between the preheparin EC-SODactivity and the activity after the second heparin injection was takenas 100% release. The difference between the preheparin activity and themean activity of the 10 and minutes samples after the first dose arepresented in relation to the "100% release". The separate experimentswere performed with intervals of at least 4 days (cf. Example 9).

FIG. 9 shows the separation of plasma on heparin-Sepharose®. Humanplasma collected before (O) and 10 minutes after intravenous injectionof heparin (200 IU/kg body weight) (Δ) was separated onheparin-Sepharose® as described in Example 9. indicates the result ofchromatography of the preheparin plasma sample after pretreatment withanti-EC-SOD-Sepharose® to remove EC-SOD. The full line representsabsorbance at 280 nm and the dotted line the NaCl gadient. EC-SODfractions A, B and C were determined in pools as indicated in thefigure. The activity in pool B and C represents EC-SOD only, since allactivity was adsorbed by the anti-EC-SOD-Sepharose®. Pool A containsalso CuZn SOD and cyanide resistant SOD activity. The EC-SOD activity infraction A was therefore determined with immobilized antibodies asdescribed for plasma in Example 9. The EC-SOD fractions A, B and C were5.2, 4.4 and 5.1 U/ml in preheparin plasma and 7.7, 5.7 and 29.4 U/mi inpostheparin plasma. The recoveries of EC-SOD activity in thechromatograms were 84% and 83%, respectively. Note that the larger totalSOD activity in pool A in the preheparin plasma chromatogram is due tohemolysis in the sample with release of CuZn SOD from erythrocytes (cf.Example 9).

FIGS. 10 and 11 are graphs showing the effect of injection of ¹²⁵¹I-labelled human EC-SOD into rabbits, and the effect of heparininjection (cf. Example 10).

FIG. 12 is a graph showing the elution pattern of recombinant EC-SODfrom monoclonal anti-EC-SOD-Sepharose® (cf. Example 17).

FIG. 13 is a graph showing the elution pattern of recombinant EC-SODfrom DEAE-Sephacel® (cf. Example 17).

FIG. 14 is a graph showing the elution pattern of recombinant EC-SODfrom heparin-Sepharose® (cf. Example 17).

FIG. 15 shows the electrophoresis of native and recombinant EC-SOD ongradient polyacrylamide gels in the presence of sodium dodecyl sulphate(cf. Example 19).

EXAMPLE1 Preparation of umbilical cord homogenates

Human umbilical cords were collected at the maternity ward of UmeaUniversity Hospital. They were kept in a refrigerator at the ward andfrozen rapidly at the laboratory at -80° C. and then stored at -30° C.before use.

After thawing, the umbilical cords were minced. They were then suspendedin 50 ml of K phosphate buffer, pH 7.4, containing 0.3M KBr, 3 mM DTPA,100,000 klU/l Trasylol®, (aprotinin) and 0.5 mMphenylmethylsulphonylfluoride (PMSF). 4 l of buffer per kg of umbilicalcord were employed. The chaotropic salt KBr was used to increaseextraction of EC-SOD from the tissue (about 3-fold). DTPA, Trasylol® andPMSF were added to inhibit proteases. The suspension was thenhomogenized in a Waring blender and finally treated with a sonicator. Itis then shaken at 4° C. for 1 hour. The resulting homogenates werecentrifuged (6000× g , 20 min.), and the supernatants were rapidlyfrozen at -80° C. and finally kept at -30° C.

EXAMPLE 2 Preparation and purification of human lung EC-SOD

Human lungs were obtained, within 24 hours after death, at autopsy fromnine patients without any apparent lung disease. The lungs were cut intopieces and excess blood was washed away in 0.15M NaCl. The pieces werehomogenized in a Waring blender in 5 volumes of ice-cold 50 mM Naacetate at pH 5.50. The homogenate was then sonicated, allowed toextract for 30 minutes at 4° C., and finally centrifuged (6,000× g) for20 minutes.

The supernatant was batch-adsorbed on DEAE-Sephacel® (obtained fromPharmacia, Uppsala, Sweden) (1 volume per 25 volumes of homogenate)equilibrated with 50 mM Na acetate at pH 5.50. The ion exchanger wasthen washed with the buffer, packed in a column, and eluted with agradient of 0-200 mM NaCl in the acetate buffer. The gradient volume was10 times the column volume.

The active fractions from the previous step were pooled, diluted with1.5 volumes of distilled water, and titrated to pH 8.4 with 1M NAOH. Thepool was again batch-adsorbed to DEAE-Sephacel® equilibrated with 175 mMTris•HCl at pH 8.4 (=1 volume of ion exchanger per 10 volumes of pool).The DEAE-Sephacel® was subsequently washed with the buffer, packed in acolumn and, eluted with 10 column volumes of a 0-200 mM NaCl gradient inthe Tris buffer.

The pooled fractions from the previous step were concentrated anddialyzed against 150 mM Na phosphate at pH 6.5. The sample was appliedto a column (about 1 ml of gel per 15 mg of protein in the sample) withPhenyl-Sepharose® (obtained from Pharmacia, Uppsala, Sweden)equilibrated against the same buffer. The activity was eluted with 20column volumes of a 0-0.5M KBr gradient in 50 mM Na phosphate at pH 6.5.

Active fractions from phenyl-Sepharose® were pooled, concentrated, anddialyzed against 0.15M Na phosphate at pH 6.5. The sample was applied toa column of Con A-Sepharose® (obtained from Pharmacia, Uppsala, Sweden)(1 ml of gel per 2 mg of protein in the sample), equilibrated againstthe phosphate buffer, and then pulse-eluted with 50 mM α-methylD-mannoside in the phosphate buffer.

Active fractions from the Con A-Sepharose® were pooled, concentrated,applied to an Ultrogel ACA -34 column (2.5×83 cm) (obtained from LKB,Stockholm, Sweden), and eluted in 50 mM Na phosphate at pH 6.5. Theelution rate was 5 ml•h⁻¹ •cm⁻².

Active fractions from the elution were pooled, concentrated, and appliedto a wheat germ lectin-Sepharose® column (10 ml) (obtained fromPharmacia, Uppsala, Sweden) equilibrated with 0.15M Na phosphate at pH6.5. The enzyme was pulse-eluted with 0.45M N-acetyl-D-glucosamine inthe phosphate buffer.

Active fractions from the above step were pooled, concentrated, dialyzedagainst 0.15M Na phosphate at pH 6.5, and applied to a blue Sepharose®CL-6B (Cibacron Blue F3G-A) column (bed volume 6 mi) (obtained fromPharmacia, Uppsala, Sweden) equilibrated with the phosphate buffer.After washing the column with buffer, a pulse of 10 mM NAD and 10 mMNADP in the buffer was introduced. After the pyridine nucleotides hadbeen washed out with buffer, the enzyme was pulse-eluted with 0.9MKBr/50 mM Na phosphate, pH 6.5.

The active fractions from the blue Sepharose® column were pooled,dialyzed against 25 mM Na phosphate at pH 6.5, and applied to aheparin-Sepharose® column (bed volume, 10 ml) (obtained from Pharmacia,Uppsala, Sweden) equilibrated with the same buffer. The column was theneluted with 140 ml of a 0-1M NaCl gradient in the phosphate buffer. Theactivity eluted in three distinct peaks: A did not bind to the heparin,B desorbed early in the gradient, and C desorbed late.

Peak A contained UV-absorbing material which probably had leaked fromthe heparin-Sepharose® column and was further purified on a Sephacryl®S-300 column (1.6×90 cm) (obtained from Pharmacia, Uppsala, Sweden). Thesample was eluted in 25 mM Tris•HCl at pH 7.5.

Fractions A, B, and C were dialyzed against 25 mM Tris•HCl at pH 7.5 andthen concentrated to about 1 ml on Amicon UM-10 ultrafiltrationmembranes. Fraction C was employed to produce EC-SOD antibodies asdescribed in the following example.

EXAMPLE 3 Preparation of rabbit-antihuman EC-SOD

EC-SOD, fraction C, was prepared as described in Example 2. A rabbit wassubcutaneously injected with 30 μg of EC-SOD in Freund's completeadjuvant. The immunization was then boostered with 5 injections of 30 μgof EC-SOD in Freund's incomplete adjuvant at intervals of one month. 2weeks after the last booster dose, the rabbit was bled to death and theserum collected. The IgG fraction of the antiserum was isolated byadsorption and desorption from Protein A-Sepharose® as recommended bythe manufacturer (Pharmacia, Uppsala, Sweden). The elution from thecolumn was performed with 0.1M glycine-HCl pH 3.0. Immediately afterelution, the pooled IgG was titrated to pH 7.0. The IgG was thereafterdialyzed against 0.1M Na carbonate, pH 8.3+0.15M NaCl, "couplingbuffer".

The CNBr-activated Sepharose® was swollen and prepared as recommended bythe manufacturer (Pharmacia, Uppsala, Sweden). The IgG as describedabove was diluted to about 5-8 mg/ml with "coupling buffer".CNBr-activated Sepharose® was added and the mixture incubated withshaking overnight at 4° C. About 5 mg IgG per mi gel was coupled. Thebuffer was sucked off from the gel and analysed for remaining protein.Over 98% coupling is generally achieved. The IgG-coupled gel was thenblocked by suspension in 1M ethanolamine overnight at 4° C. Then the gelwas washed with "coupling buffer", followed by 0.1M Na acetate, pH4.0+0.5M NaCl. The gel was then kept in "coupling buffer" with azide asantibacterial agent.

The maximum binding capacity of the immobilized antibodies wasdetermined by incubation overnight with an excess of EC-SOD and analysisof the remaining EC-SOD. After centrifugation the result was comparedwith a sham incubation with Sepharose® 4B.

100 μl of a 50% suspension of anti-EC-SOD-Sepharose was added to 0.5 mlEC-SOD in "coupling buffer". A parallel sham incubation with 100 μl of a500b suspension of Sepharose® 4B was performed. The solutions wereshaken overnight at 4° C. and then centrifuged. The remaining activityin the Sepharose® 4B-treated solution was 2080 U/ml and in theanti-EC-SOD-Sepharose-treated solution it was 720 Units/ml. Using thesefigures it could be calculated that 1 ml of anti-EC-SOD-Sepharose gelbound 13500 units of EC-SOD (about 120 μg). This figure was used for theplanning of the adsorption of EC-SOD from human tissue homogenates.

EXAMPLE 4 Immunoadsorption of EC-SOD to anti-EC-SOD-Sepharose®

10 l of umbilical cord extract prepared as described in Example 1 washandled at a time. The EC-SOD content of the extract was about 150 U/ml.If the gel binds 13,600 U/mi (cf. Example 3), adsorption of all EC-SODin 10 I of extract required about 110 ml of anti-EC-SOD-Sepharose.

First the extract is centrifuged (6000× g, 30 min.) to removeprecipitated protein. To the supernatant w then added 110 ml ofanti-EC-SOD-Sepharose, and the mixture was incubated overnight at 4° C.with stirring. The gel was separated from the extract on a glass funnel.The gel was then washed on the funnel with large amounts of 50 mM Kphosphate, pH 7.0+0.5M NaCl.

The gel was packed in a chromatography column with a diameter of 5 cm.The elution started with 50 mM K phosphate, pH 7.0+0.5M NaCl at a rateof 50 ml/hr and the absorbance at 280 nm is recorded. The elution wascontinued until a very low A₂₈₀ is attained. The EC-SOD was then elutedwith a linear gradient of KSCN, 0.5-2.5M, in 50 mM K phosphate, pH 7.0.The total volume of the gradient is 500 ml, and the elution is run at 30ml/hr. The desorption of EC-SOD was slow and the elution could not bespeeded up.

The eluting fluid was collected in a fraction collector. To protecteluted EC-SOD from the high KSCN concentration, a T-pipe was insertedinto the plastic tube from the eluting end of the column and distilledwater injected at twice the rate of the column elution.

The resulting elution of protein (at A₂₈₀ and EC-SOD is shown in FIG. 1.It appears that the elution of EC-SOD is not complete at the end of thegradient 2.5M KSCN, but the elution is not continued in order not tocollect EC-SOD which has become too denatured by the highKSCN-concentration.

The EC-SOD was pooled as shown in the figure. The first activity in thegradient were not collected since it contained a rather large amount ofcontaminating, unspecifically bound protein (A₂₈₀ ). Most of the A₂₈₀ inthe gradient is contributed by the KSCN, and only in the beginning is asignificant small protein peak seen. For regeneration the gel is thenshaken overnight in the 2.5M KSN, then washed with 50 mM K phosphate, pH6.5+0.5M NaCl and then with buffer without any NaCl. Azide is finallyadded as an antibacterial agent. Before use the gel is washed withazide-free 50 mM K phosphate pH 6.5.

EXAMPLE 5 Adsorption to and elution from DEAE-Sephacel®

To the pooled eluate from the anti-EC-SOD-Sepharose column was added1-aminomethylpropanol to a final concentration of 10 mM. The solutionwas then titrated to pH 9.0 with 1M NAOH. The solution was diluted withabout 5 volumes of distilled water. To the resulting solution was added40 ml DEAE-Sephacel® equilibrated with 50 mM Na phosphate+0.5M NaCl+175mM Tris-HCl pH 9.6.

The EC-SOD was allowed to adsorb to the DEAE-Sephacel® with stirringovernight at 4° C. The DEAE-Sephacel® was then collected on aglass-funnel, washed with 50 mM Na phosphate, pH 6.5, and packed in achromatography column with a diameter of 2.5 cm. The column was firsteluted with about 4 volumes of the above buffer. The EC-SOD was theneluted with 50 mM Na phosphate pH 6.5+0.25M NaCl as shown in FIG. 2. Theactivity was pooled as shown in FIG. 2, dialyzed against 25 mM Naphosphate pH 6.5, and finally concentrated to about 2 ml.

EXAMPLE 6 Final purification of EC-SOD on heparin-Sepharose®

Four batches of eluate from the DEAE-Sephacel®, as described above, wereseparated at a time on heparin-Sepharose®. The enzyme solutions to beapplied were dialyzed against 25 mM K phosphate pH 6.5.

20 ml heparin-Sepharose® gel was prepared as recommended by themanufacturer (Pharmacia, Uppsala, Sweden), and washed with 25 mM Kphosphate pH 6.5 containing 1M NaCl and then with buffer without NaCl.The heparin-Sepharose® was packed in a chromatography column with adiameter of 2.5 cm, and elution was started with 25 mM K phosphate pH6.5 at 15 ml/hr. The EC-SOD solution (about 10 ml, 800,000 units) wasthen applied and the absorbance at 280 nm monitored (cf. FIG. 3). When,after the first peak, the A₂₈₀ approached the base-line, the EC-SOD waseluted with an NaCl gradient from 0 to 1.2M NaCl. The gradient volumewas 400 ml. The EC-SOD eluted in three peaks; one with no affinity forheparin, one with intermediate affinity and one with high affinity. Thepeaks correspond to the fractions A, B and C described in Example 2.When purified by the present procedure, almost all activity is of typeC, and it was therefore concluded that this is likely to be the nativeform of the enzyme. Peak C was pooled as shown in FIG. 3.

The pooled activity was 430,000 units (about 4.3 mg). The specificactivity was 81100 (U per ml/A₂₈₀). On SDS-PAGE gel subunits of about30.000 D were found. No trace of contamination was seen. The pooledactivity respresented about 53% of the activity applied on theheparin-Sepharose®.

EXAMPLE 7 The amino-terminal sequence of human EC-SOD

Human EC-SOD obtained as described in the preceding Examples wasanalysed for its (N-terminal) amino acid sequence by standard procedures(Edman et al., Eur. J. Biochem. 1, 1967, pp. 80-87). The sequence of thefirst 33 amino acids was found to be: ##STR9##

This sequence--or a suitable part of it--may be employed to producesynthetic DNA probes, synthetic deoxyoligonucleotides complementary toboth the coding and non-coding strand of the DNA sequence coding for theamino acid sequence shown above.

Such probes may be used in hybridization experiments with cDNA librariesproduced from MRNA from EC-SOD-producing cells or tissues, in order toisolate a full-length or partial cDNA copy of an EC-SOD gene, asdescribed in the following Example.

EXAMPLE 8 Cloning and Sequencing of human EC-SOD Preparation of a DNAprobe

Human EC-SOD was purified from umbilical cords substantially asdescribed in Examples 1-6 above, and the sequence of the first 33N-terminal amino acids was determined as described in Example 7. On thebasis of this amino acid sequence and the postulated codon usage(Grantham et al., Nucl. Acids Res. 9, 1981, pp. 43-74) for eukaryoticproteins, a synthetic 48-meric deoxyoligonucleotide ##STR10##complementary to the coding strand of the EC-SOD gene was synthetizedaccording to the procedure described by Matthes et al. (EMBO Journal 3,1984, pp. 801-805).

Screening of a human placenta cDNA library

A human placenta cDNA library prepared in the vector λgtll was obtainedfrom Clontech Laboratories, Inc., 922 Industrial Avenue, Palo Alto,Calif. 94303, USA (Catalogue No. HL 1008 Lot No. 1205). The recombinantphases were screened for human EC-SOD cDNA sequences by plating thephases on the indicator strain E. coli Y 1090. Transfer of plaques toand treatment of nitrocellulose filters (Hybond C, Amersham Inc.) wereessentially as described by Maniatis et al. (Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratories, 1982). Eightnitrocelfulose filters to which 20,000 plaques had been transferred perfilter, were presoaked in 5×SSC and then prehybridized for one hour at41° C. in 40 ml of 20% formamide, 5×Denhardt's solution, 50 mM sodiumphosphate, pH 6.8, and 50 μg/ml of denatured sonicated calf thymus DNA.The filters were then hybridized to 7×10⁵ counts per minute per ml ofthe ³² P-γ-ATP end-labelled (cf. Maniatis et al., op.cit.) 48-mericprobe described above. The hybridization was performed in theprehybridization solution supplemented with 100 μM ATP (finalconcentration) for 18 hours at 41° C. After incubation overnight at 41°C., the filters were washed once in 0.2× SSC at 37° C. followed by 4washes in 0.2× SSC, 0.1% SDS, at 37° C. and were then allowed toair-dry. The filters were exposed to DuPont Cronex 4 X-ray filmovernight. FIG. 4 shows one of the autoradiograms. Each filter containedabout 6 positive plaques.

Phages from plaques showing a positive hybridization reaction wereisolated and purified, and DNA from these phases was extracted by themethods described by Davis et al. (Advanced Bacterial Genetics, ColdSpring Harbor Laboratories, 1980), and the length of the cDNA insertswas determined by agarose gel electrophoresis after cleavage of thephage DNA with the restriction endonuclease EcoRl. The recombinant phagecarrying the longest cDNA insert was designated λSP3 and was chosen forfurther studies.

The cDNA insert from phage λELP3 was subjected to restrictionendonuclease analysis and sequenced after subcloning into pUC18 and theM13 vector mp9, respectively. The insert was demonstrated to be DNAencoding human EC-SOD by comparing the amino acid sequence derived fromthe DNA sequence with the peptide sequence of the purified protein andby its expression product in CHO cells. The insert isolated from λSP3contained 1396 bp of cDNA and an open reading frame encoding a proteinof 240 amino acids and a 69 bp 5' untranslated region and a 607 bp 3'untranslated region. (See FIGS. 5A and B)

Subcloning and restriction endonuclease analysis of cDNA insertsencoding human EC-SOD

About 30 μg of λSP3 DNA were digested with the restriction endonucleaseEcoRl, and the cDNA insert was separated from λ DNA by electrophoresisin a 6% polyacrylamide gel. Approximately 0.2 μg of the cDNA fragmentwas isolated from the gel by electroelution, phenol and chloroformextraction and ethanol precipitation (Maniatis et al., supra). 0.05 μgof the isolated cDNA fragment was ligated to 1 μg of restrictionendonuclease EcoRl-digested alkaline phosphatase treated pUC18 DNA(Norrander et al., Gene 26, 1983, pp. 101-106). The ligated DNA wastransformed to strain E. coli HB101 (J. Mol. Biol. 41, p. 459).Transformants were selected on plates containing ampicillin. Arecombinant plasmid carrying the cDNA insert was identified anddesignated pLS3. Plasmid pLS3 DNA was subjected to restrictionendonuclease analysis.

DNA sequence analysis of cDNA encoding human EC-SOD

The DNA sequence of the cDNA insert from phage λSP3 encoding humanEC-SOD was determined by the procedures of Sanger et al. (Proc. Natl.Acad. Sci. USA 74, 1977, pp. 5463-5467; F. Sanger and A. R. Coulson,FEBS Letters 87, 1978, pp. 107-110) and Messing et al. (Nucl. Acids Res.9, 1981, pp. 309-321; P. H. Schreir and R. Cortese, J. Mol. Biol. 129,1979, pp. 169-172) after cloning of the cDNA fragment into EcoRl sitethe M13 vector mp9 (J. Messing and J. Vieira, Gene 19, 1982, pp.269-376).

A sequential series of overlapping clones of the cDNA insert wasgenerated in the M13 vector mp9 according to the method described byDale et al. (Plasmid 13, 1985, pp. 31-40). Using this method thecomplete nucleotide sequence of both DN strands of the cDNA wasdetermined and was found to have a length of 1396 bp.

The nucleotide sequence and deduced amino acid sequences of the cDNAinsert of λPS3 are shown in FIG. 5. The cDNA insert has an open readingframe of 240 amino acids. The amino acid sequence of the purified matureprotein is initiated at amino acid +1 which suggests a putative signalpeptide of 18 amino acids. Like known signal peptides, this sequence ofamino acids is rich in hydrophobic amino acids and moreover, the lastresidue is alanin which is one of the amino acids found in this positionin known signal peptides (G. Von Heijne, Eur. J. Biochem. 133, 1983, pp.17-21). Underlined amino acid sequences have been confirmed by peptidesequencing.

Another feature of the DNA sequence is the sequence found at thetranslation initiation codon, --CAGCCAUGC--, which is homologous to thepostulated consensus sequence - for eukaryotic initiation sites,--CC_(G) ^(A) CC--AUG(G)-- (M. Kozak, Nucl. Acids Res. 12, 1984, pp.857-872). Moreover, a possible polyadenylation signal with the sequence--ATTAAA-- homologous to the postulated consensus sequence AATAAA isfound 14 bp upstream of the polyadenylation tail.

Expression of human EC-SOD in CHO cells

An expression vector containing the Simian Virus 40 (SV40) origin ofreplication, early and late promoters, polyadenylation and terminationsequences was used to produce human EC-SOD encoded by the cDNA describedabove. The 1396 bp long cDNA was inserted into a unique EcoRlrestriction endonuclease cleavage site located between the SV40 earlypromoter and the SV40 polyadenylation and termination sequences so thatthe expression of the coding sequence for human EC-SOD is controlled bythe SV40 early promoter (FIG. 6). This constructed EC-SOD expressionplasmid was designated pPS3.

20 μg of pPS3 DNA were linearized by cleavage with the restrictionendonuclease Pstl at the unique Psti site located in the β-lactamasegene. The linearized pPS3 DNA was co-transfected with 0.5 μg of DNA froma plasmid containing sequences conferring resistance to Geniticin (G-418sulphate, Gibco Ltd.) into CHO-K1 (ATCC CCL61) cells by the method ofGraham and Van der Eb (Virology 52, 1973, pp. 456-467). Transfectedcells were selected by growth in medium (Hams's P12 medium supplementedwith 10% fetal calf serum, streptomycin and penicillin) containing 700μg per ml of Geniticin (G-418 sulphate, Gibco Ltd.). Geniticin resistantcolonies were isolated and propagated in the same medium. Medium wasremoved at intervals and assayed for the presence of EC-SOD by ELISA andenzyme activity measurements as described below.

Several of the cell lines tested showed comparable production of humanEC-SOD and one of the obtained cell lines was denoted CHO-K1/pPS3neo-18and selected for further studies.

Secretion of EC-SOD into the culture medium by CHO cells containing thegene encoding human EC-SOD

The clone CHO-K1/pPS3neo-18 and the parental CHO-K1cells were grown toconfluency in Ham's F-12 medium containing 10% of fetal calf serum.After 3 additional days the medium was removed and the cells washedtwice with phosphate buffered saline. The cells were then detached fromthe culture flasks by means of incubation in a solution containing 40 mMTris-HCl, 140 mM NaCl and 1 mM EDTA. The recovered cells (about 8×10⁶)were then centrifuged, the supernatant discarded and the cells stored at-80° C. as a pellet. The cells were then disintegrated with sonicationin 1.5 ml of a solution containing 50 mM K phosphate pH 7.4, 0.3M KBr, 3mM DTPA, 0.5 mM PMSF and 100 KIE/ml trasylol. The homogenates werecentrifuged. Specific determination of the amount of EC-SOD wasperformed by means of incubation of the homogenates and culture mediawith immobilized antibodies directed towards human EC-SOD and human CuZnSOD, as outlined in Ohman and Marklund, Chim. Sci. 70, 1986, pp.365-369. No EC-SOD was found in the parental CHO K1 cells or in theirculture medium. The culture medium from the clone CHO-K1(pPS3neo-18 (15ml) cells was found in this particular experiment to contain 51 U/mlEC-SOD, total 765 U. The cell homogenate (in 1.5 ml) contain 71 U of SODactivity of which 20 U was human EC-SOD. Thus, 97.5% of the EC-SOD inthe CHO-K1/pPS3neo-18 culture was secreted into the medium.

Production of human EC-SOD in CHO cells

The production of human EC-SOD by this clone was determined both whenthe cells were grown on a solid support and in suspension.

1. Production of EC-SOD by CHO-K1/pPS3neo-18 cells growing on solidsupports

a) A 175 cm³ flask was inoculated with 4.5×10⁶ cells in 30 ml of Ham'sF12 medium (Flow Laboratories) supplemented with 10% fetal calf serum, 2mM L-glutamine, streptomycin and penicillin. The cells were incubated at37° C. in air containing 5% CO₂. Medium was changed every third day, andthe concentration of human EC-SOD secreted into the growth medium wasdetermined as described below in the section entitled "Assays fordetection of the expression of human EC-SOD". The productivity of humanEC-SOD was 1.5 pg.cell⁻¹. 24 hours⁻¹ as measured by ELISA and bydetermining the EC-SOD enzyme activity.

b) Microcarriers (m.c) (Cytodex 3, Pharmacia, Sweden), 4 mg/ml, wereinoculated with cells at a concentration of 7 cells/m.c in Ham's F12medium (Flow Laboratories) supplemented with 5% fetal calf serum, 2 mML-glutamine, streptomycin and penicillin.

The cells were grown in a 500 ml stirrer flask (Techne) at 37° C. in 5%CO₂ in air. At confluent cell growth, the culture was perfundated at arate of 0.088.h⁻¹.

The productivity of human EC-SOD was 0.50 pg.cell⁻¹. 24 hours⁻¹.

2. Production of EC-SOD by CHO-K1/pPS3neo-18 cells grown in suspensionculture

125 ml of medium (Ham's F12 supplemented with 1006 fetal calf serum, 2mM L-glutamine, streptomycin and penicillin) were inoculated to 2×10⁵cells/mi. The culture was incubated in spinner flasks (Techne) at 37° C.in air containing 5% CO₂.

Every third day medium was changed. Productivity of human EC-SOD was0.65 pg.cell⁻¹. 24 hours⁻¹.

Assays for the detection of expression of human EC-SOD 1. Enzyme linkedimmunoadsorbent assay (ELISA)

Microtiter plates (Nuncion, NUNC A/S, Denmark) were coated with 100 μlper well of a solution containing 15 μg per ml of polyclonal rabbitanti-EC-SOD IgG antibodies (prepared as described in Example 3) in 15 mMNa₂ CO₃, 35 mM NAHCO₃, 0.02% NaN₃, pH 9.6. After incubation overnight atroom temperature, the plates were washed with PBS (10 mM sodiumphosphatia, 145 mM NaCl, pH 7.2). The microtiter plates were incubatedfor 30 minutes at 37° C. with 200 μl per well of a solution containing3% (w/v) bovine serum albumin in PBS and washed with PBS. 100 μl ofdiluted medium samples were added to each well and incubated for 1 hourat 37° C. The plates were washed with 5% Tween® 20 (v/v) in PBS followedby incubation for 1 hour at 37° C. with 100 μl per well of a solutioncontaining 8 μg per ml of a mouse monoclonal anti-EC-SOD antibody (seeExample Z) in 3% bovine serum albumin in PBS. After washing with 5%Tween® 20 in PBS, the plates were incubated for 1 hour at 37° C. with aperoxidase-conjugated rabbit anti-mouse antibody (Dakopatts A/S,Denmark) in 3% bovine serum albumin in PBS. The plates were washed with5% Tweens® 20 in PBS and incubated for 20 minutes at room temperature inthe dark with 100 ill per well of a substrate solution (50 mM sodiumcitrate, 100 mM sodium phosphate, 0.04% (w/v) o-phenylene diamine, 0.01%H₂ O₂, pH 5.0). The reaction was stopped by adding 25 μl of 10% SDS perwell and the absorbance at 450 nm measured.

2. Determination of EC-SOD enzyme activity

SOD enzyme activity was determined as described in Marklund, J. Biol.Chem. 251, 1976, pp. 7504-7507. To achieve specificity for human EC-SODthe samples were analysed before and after treatment with anti-humanEC-SOD immobilized on Sepharose® (see Example 4). EC-SOD activity wastaken as the difference between the activity of the sample before andafter adsorption to the antibody.

EXAMPLE 9 Heparin-induced release of EC-SOD into human blood plasma

200 IU/kg body weight of heparin (obtained from AB KABI-Vitrum,Stockholm, Sweden) were injected intravenously into two healthy malesfasted overnight [a) 34 years of age and b) 40 years of age]. Bloodsamples were taken before heparin injection and at intervals after theinjection as indicated in FIG. 7. The blood samples were tapped intoTerumo Venoject vacuum tubes containing EDTA as anticoagulant andcentrifuged. After centrifugation, the plasma samples were kept at -80°C. until assay.

Furthermore, 20 ml of whole blood were taken from three healthy personsand kept in EDTA tubes as described above. The blood was divided intotwo equal parts and to one was added 30 IU heparin/ml and to the otheran equal volume of 0.15M NaCl. After incubation for 30 minutes at roomtemperature, the samples were centrifuged and the plasma collected forSOD assay.

SOD activity was assayed by means of the direct spectrophotometricmethod employing KO₂ (S. L. Marklund, J. Biol. Chem. 251, 1976, pp.7504-7507) with modifications as described in "Ohmann and Marklund,Clin. Sci. 70, 1986, pp. 365-369. One unit of SOD activity correspondsto 8.3 ng human CuZnSOD, 8.8 ng human EC-SOD and 65 ng bovine MNSOD.Distinction between isoenzymes in plasma was achieved by means ofantibodies towards human CuZnSOD and EC-SOD immobilized on Sepharose® 4Bas described in Ohmann and Marklund, Clin. Sci. 70, 1986, pp. 365-369.

The results are shown in FIG. 7 indicating that an intravenous injectionof 200 IU heparin per kg body weight leads to a rapid three-fold rise inplasma EC-SOD activity. The maximum increase is approached already after5 minutes. The activity stays high for 15-30 minutes and then decreasesgradually to approach the initial level after more than 6 hours.Intravenously injected heparin had no effect on the plasma CuZnSOD andcyanide resistant SOD activities.

The effect of administering up to 200 IU/kg body weight of intravenousheparin on the release of EC-SOD to plasma is shown in FIG. 8. Itappears from the figures that increasing doses of heparin result in anincreased release of EC-SOD. Apparently no distinct plateau is reachedand it is likely that doses over 200 IU/kg body weight would result inan even higher EC-SOD release. Ethical considerations, however,precluded testing of higher doses.

Contrary to the results obtained in vivo, addition of heparin to wholeblood as described had no effect on the plasma EC-SOD activity. Nor didaddition of heparin (5 IU/mi) -directly to plasma result in any changein the EC-SOD activity. The results indicate that the increase in plasmaEC-SOD activity in vivo as seen in FIG. 7 is not caused by any releaseof the enzyme from blood cells, or by activation of the EC-SOD presentin plasma.

Plasma samples from 5 healthy persons (3 males, 2 females) weresubjected to chromatography on heparin-Sepharose® (purchased fromPharmacia, Uppsala, Sweden). Chromatography was carried out at roomtemperature in columns containing 2 ml of heparin-Sepharose® with 25 mMpotassium phosphate, pH 6.5, as eluant. The samples (2 ml plasma) wereapplied at 4.2 ml/h and when the A₂₈₀ approached baseline, boundcomponents were eluted with a linear NaCl gradient in the potassiumphosphate buffer (0-1M, total volume 50 ml) at 9 ml/h (cf. FIG. 9). Themean yield of SOD activity in the eluate was about 95%.

Before application, the plasma samples were equilibrated with theelution buffer by means of chromatography on small Sephadex® G 15columns (PD-10) (also purchased from Pharmacia, Uppsala, Sweden). Thechromatography resulted in a three-fold dilution of the samples. Therecovery of SOD activity was close to 100%.

The results of the determination of EC-SOD fractions A, B and C in fivenormal plasma specimens are shown in Table I below. It was found thatthe three fractions are roughly equally large in normal plasma. The meanyield of EC-SOD activity in the chromatogram was 95%. The separationinto three fractions is apparently not caused by secondary in vitrodegradation, since the patterns for a plasma specimen was identicalbefore and after storage for 3 days in a refrigerator. The effect ofintravenous heparin on the composition of EC-SOD fractions in plasma isshown in FIG. 9. It was found that intravenous injection of heparin inthe person analysed leads to a significant increase in fraction C only.A and B remain essentially unchanged. In a second analysed person (datanot shown), the effect of heparin injection was essentially identical.Fraction C increased from 7 to 32 units/ml plasma.

                  TABLE I                                                         ______________________________________                                        Separation of plasma EC-SOD into fractions A, B and C                                  EC-SOD, U/ml plasma                                                           Fractions                                                            Age/sex    A           B        C                                             ______________________________________                                        40/male    5.9         6.2      7.0                                           34/male    5.2         4.4      5.1                                           32/male    2.3         5.2      6.4                                           33/female  2.3         5.9      8.3                                           29/female  3.3         6.1      7.3                                           mean ± SD                                                                             3.5 ± 1.5                                                                              5.6 ± 0.8                                                                           6.8 ± 1.2                                  ______________________________________                                    

The experiments described above show that intravenous injection ofheparin leads to a prompt increase in plasma EC-SOD activity. Heparindoes not activate EC-SOD, nor can any release from blood cells bedemonstrated, pointing to the endothelial cell surfaces as the mostlikely source of the released EC-SOD. A number of other factors withaffinity for heparin, lipoprotein lipase, hepatic lipase, diamineoxidase, and platelet factor 4 have previously similarly been shown tobe rapidly released by intravenous heparin. In most of these cases,there is evidence that heparin-induced displacement of the protein fromheparan sulfate on endothelial cell surfaces is the explanation of thephenomenon (A. Robinson-White et al., J. Clin. Invest. 76, 1985, pp.93-100; C. Busch et al., Throm. Res. 19, 1980, pp. 129-137). It islikely that the release of EC-SOD can be explained in the same way.

No distinct plateau in the release was reached for heparin doses up to200 IU/kg body weight, showing that more heparin is needed for maximumrelease of EC-SOD than for lipoprotein lipase, diamine oxidase, hepaticlipase, and platelet factor 4. The ratio between the affinity forheparin and heparan sulfate might be lower for EC-SOD than for the otherproteins.

Basal human plasma contains nearly equal amounts of EC-SOD fraction A, Band C. Intravenous heparin released only the high-heparin affinityfraction C, which is apparently the form which has affinity forendothelial cell surfaces. The increase achieved here was 4-6-fold, butit is likely that higher doses of heparin would result in a higherratio. Much higher ratios are achieved for lipoprotein lipase, hepaticlipase, diamine oxidase, and platelet factor 4. Compared with theseproteins, the endothelial binding of EC-SOD appears rather loose.Possibly an equilibrium exists for EC-SOD fraction C between plasma andendothelial cell surfaces. Most EC-SOD in the vascular system appears tobe located on the endothelial cell surfaces.

The molecular background for the difference in heparin affinity betweenEC-SOD fraction A, B and C is still unresolved. The amino acid andsubunit compositions were not significantly different (S. L. Marklund,Proc. Natl. Acad. Sci. USA 79, 1982, pp. 7634-7638). Nor could anyantigenic differences be detected (S. L. Marklund, Biochem. J. 220,1984, pp. 269-272). The binding to negatively charged heparin isapparently not of a general ion-exchange nature since no differencebetween fraction A and C can be detected upon ion exchangechromatography, and their isoelectric points are identical (pH 4.5). Thedifference is not due to in vitro degradation, since storage of plasmafor 3 days in a refrigerator did not change the elution pattern onheparin-Sepharose®. Although in vivo degradation is a possibility, onemight speculate that fractions A and B are specifically intended forprotection of fluid components and fraction C for shielding cellularsurfaces.

Most cell types in the body possess heparan sulfate and other sulfatedglucoseaminoglycanes on their surfaces (M. Hook et al., Ann. Rev.Biochem., 53, 1984, pp. 847-869). It is possible that much of the EC-SODfound in tissues (S. L. Marklund, J. Clin. Invest. 74, 1984, pp.1398-1403) is located on such substances on cell membranes and in theconnective tissue. The binding of EC-SOD to cellular surfaces might bean especially efficient way of protecting cells against extracellularlyformed superoxide radicals. It is interesting to note that substitutionof CuZnSOD with polylysine to facilitate association with negativelycharged cell membranes highly potentiated the ability of the enzyme toprotect activated polymorphonuclear leukocytes against self-inactivation(M. L. Salin and J. M. McCord, in Superoxide and Superoxide Dismutases,eds. A. M. Michelson, J. M. McCord and I. Fridovich, Academic Press,1977, pp. 257-270). The cell membrane-associated SOD of Nocardiaasteroides confers efficient protection to the bacterium againstactivated polymorphonuclear leukocytes (B. L. Beaman et al., Infect.Immun. 47, 1985, pp. 135-140). Microorganisms lacking affinity forEC-SOD fraction C would, unlike most cells in the body, not benefit fromprotection by the enzyme.

There is evidence that superoxide radicals produced by activatedleukocytes and also by other cell types under certain conditions can,directly or indirectly, induce chromosomal damage (J. Emerit,Lymphokines 8, 1983, pp. 413-424; A. B. Weitberg, S. A. Weitzman, E. P.Clark and T. P. Stossel, J. Clin. Invest. 75, 1985, pp. 1835-1841; H. C.Birnboim and M. Kanabus-Kaminska, Proc. Natl. Acad. Sci. USA 82, 1985,pp. 6820-6824) and promote carcinogenesis (C. Borek and W. Troll, Proc.Natl. Acad. Sci. USA 80, 1983, pp. 1304-1307; Y. Nakamura, N. H. Colburnand T. D. Gindhart, Carcinogenesis 6, 1985, pp. 229-235). Thesurface-associated EC-SOD fraction C would be an efficient protectoragainst such events in vivo. In most in vitro test systems, much of theEC-SOD fraction C would probably be lost from the cells since thebinding appears to be weak. Findings in such systems are then notnecessarily quantitatively predictive for the in vivo protection againstdamage.

Parenteral CuZnSOD has been shown to possess many interestingtherapeutic properties as indicated above. The present findings suggestthat administration of EC-SOD may be an even more efficient mode ofprotection against cellular damage caused by superoxide radicals inextracellular space.

EXAMPLE 10 Injection of ¹²⁵ I-labelled human EC-SOD into rabbits

Umbilical cord EC-SOD prepared as described in Examples 1 and 4-6 waslabelled with ¹²⁵ I using the "lodogen" technique (P. R. P. Salacinski,C. McLean, J. E. C. Sykes, U. V. Clement-Jones and P. J. Lowry, Anal.Biochem. 117, 1981, pp. 136-146). The labelled EC-SOD was separated from¹²⁵ I-iodide by means of gel filtration on Sephacryl® S-300 (obtainedfrom Pharmacia, Uppsala, Sweden). The location on the chromatogram wasat the same site as unlabelled EC-SOD, which indicates that themolecular size had not changed.

The resulting labelled EC-SOD was chromatographed on heparin-Sepharose®(as described above). Only material with high heparin affinity(=fraction C) was used for further experiments.

The labelled EC-SOD was injected intraveneously into rabbits (weighingabout 3 kg). Blood samples were then taken to analyse the radioactivityremaining in the plasma. The plasma samples were precipitated withtrichloroacetic acid (which precipitates proteins) and the radioactivitywas counted on the protein pellets after centrifugation. This eliminatescounting of radioactive iodide and iodine-containing amino acids in theplasma, derived from degraded EC-SOD. The rabbits were given iodide intheir drinking water to prevent relabelling with ²⁵ I of proteins invivo.

After different times, heparin (2500 IU) was injected intraveneouslyinto the rabbits, to study the effect of heparin. The results are shownin FIG. 10 and FIG. 11. 100% corresponds to the radioactivity that theplasma should theoretically contain, given the amount injected, andassuming that the total plasma volume in the rabbits were 5% of theirbody weight (e.g. 3 kg rabbit - 150 ml of plasma).

FIG. 10 shows the results when heparin is injected before, and 2, 5, 10and 20 minutes after ¹²⁵ I-EC-SOD.

After injection of labelled EC-SOD, a rapid decline in activity occurswithin 5-10 minutes to about 15% of the theoretical maximum. Whenheparin is injected before EC-SOD, almost all the EC-SOD activityremains, with only a slow decline. When heparin is injected 2, 5, 10 and20 minutes after the ¹²⁵ I-EC-SOD, there is a rapid increase inradioactivity and the peak reaches the theoretical maximum.

FIG. 11 shows injection of heparin after 2 hours to 72 hours. It appearsfrom the figure that there is a rapid increase in activity, which, after2 hours, reaches about 5CP6 of the theoretical maximum and after 72hours still reaches 3%.

There appears to be a rather rapid decline to 50% (at 2 hours), butafter that the EC-SOD is eliminated far more slowly. Using the maxima inFIG. 11, a half-life (t 1/2) of about 18 hours can be calculated. It isprobably longer in man, as turnover in the body of almost all componentsis faster in small animals.

The rapid increase in plasma EC-SOD (maximum reached within 2 minutes)after i.v. heparin indicates that the released ¹²⁵ I-EC-SOD waslocalized on the blood vessel endothelium which points to the sameconclusion as in Example 8.

EXAMPLE 11 Binding of human EC-SOD C to pig aorta endothelium

Pig aortas were collected at a slaughterhouse, kept on ice duringtransport, opened along the length and put between two 1.5 cm thickperspex blocks. 10 mm diameter holes had been drilled into the blockpositioned above the aorta facing the luminal side. Thus, 10 mm diameterwells with aorta endothelium in the bottom were achieved. A solutioncontaining 440,000 cpm ¹²⁵ I-EC-SOD (labelled as described in Example10) and a large excess of unlabelled EC-SOD fraction C (121 μg/ml) wasprepared. The solvent was Eagle's minimal essential medium buffered atPH 7.4 with HEPES and containing 0.5 μg/ml bovine serum albumin. 150 μlof the solution were put in each of three wells and incubated withshaking for 2.5 hours at room temperature. The solution was then suckedoff. The wells were then washed twice with 500 μl of solvent (2 minuteswith shaking) and then twice with 500 μl of solvent containing 15 IU ofheparin (5 minutes with shaking). The radioactivity in the solutions(mean of three wells, % of added activity) was 80.4% (incubated initialsolution), 2.1%, 0.6% (wash solutions), 6.5%, 2.2% (wash with heparin).The SOD enzyme activity was determined in solutions from one well andwas found to be 84.2% (82.7%) in the incubated initial solution removedand 7.1% (7.0,,6) in the first heparin wash (corresponding data forradioactivity in brackets). Thus, there was a very good correspondencebetween SOD enzyme activity and the radioactivity determined. The datashow that about 20% of added EC-SOD were bound by the aorta endothelium,and that EC-SOD activity could be released by the addition of heparin.

EXAMPLE 12 Binding of native and recombinant EC-SOD to lectins

Concanavalin Al, lentil lectin and wheat germ lectin immobilized onSepharose® was obtained from Pharmacia AB, Uppsala, Sweden. 2 ml of eachgel were packed in chromatography columns. 50 mM sodium phosphate (pH7.4) +0.25M NaCl was used as eluant. 200 units (1.7 μg) native umbilicalcord EC-SOD or recombinant EC-SOD dissolved in 0.5 ml elution bufferwere applied to the lectin columns. 3.5 ml elution buffer was thenapplied. The columns were washed with 10 ml elution buffer. Bound EC-SODwas then eluted with 14 ml 0.5M α-methylmannoside (ConA Sepharose® andlentil lectin-Sepharose®) on 0.5M N-acetylglucoseamine (wheat germlectin-Sepharose®). SOD activity was determined on fluid eluting fromthe columns.

98% of the native EC-SOD and 97% of recombinant EC-SOD bound to theconcanavalin A-Sepharose®. 99% of the native EC-SOD, 96% of therecombinant EC-SOD bound to lentil lectin Sepharose®. 61% of the nativeEC-SOD and 95% of the recombinant EC-SOD bound to wheat germ lectinSepharose®.

The affinity for ioncanavalin A and lentil lectin shows that both nativeand recombinant EC-SOD contain glucosyl and mannosyl residues in theircarbohydrate moieties. Affinity for wheat germ lectin indicates thepresence of N-acetyl-glucoseaminyl residues. The heterogeneity of nativeEC-SOD with regard to binding to wheat germ lectin is probably explainedby partial degradation of the carbohydrate part of the enzyme whenpresent within the umbilical cord or within the umbilical cordhomogenate during isolation. There is less risk that the recombinantenzyme is exposed to degrading enzymes. To conclude, as deduced from theresults of these studies with lectin, the carbohydrate parts of nativeand recombinant EC-SOD are similar.

EXAMPLE13 Analysis of native umbilical cord EC-SOD and recombinantEC-SOD on heparin-Sepharose®

About 500 units (4.4 μg) native EC-SOD C and recombinant EC-SOD werechromatographed on heparin-Sepharose® as described in Example 9. Bothenzymes were found to elute at 0.52M in the NaCl gradient. Thus, nativeand recombinant EC-SOD behaved identically, and the result establishesthat the recombinant EC-SOD is of the C-type.

EXAMPLE14 Content of copper and zinc in the EC-SOD molecule

The content of Cu and Zn in native umbilical cord EC-SOD and ofrecombinant EC-SOD was determined by means of atomic absorptionspectrometry in a graphite furnace in a Perkin-Elmer Zeeman 303+HGAapparatus. The amount of EC-SOD protein and Cu and Zn in thepreparations were compared. One mole of native EC-SOD (tetramer) wasfound to contain 3.97 moles of Cu and 4.50 moles of Zn. The recombinantEC-SOD contained 3.98 moles of Cu and 4.45 moles of Zn per mole enzyme.The two preparations thus contain equal amounts of Cu and Zn. Theresults confirm the previous finding of 4 moles of Cu per mole of EC-SOD(Marklund, Proc. Natl. Acad. Sci. USA 79, 1982, pp. 7634-7638). Aboutfour Zn atoms were also found in that investigation, but the presence ofzinc in the enzyme could not be established with certainty due to thescarcity of material and the possibility of Zn contamination. Thepresent results now establish that the EC-SOD molecule contains four Znatoms.

EXAMPLE15 Preparation of monoclonal antibodies against human EC-SOD

Mice were injected with EC-SOD C prepared from umbilical cord (Example4-6). After a few months the mice were injected with EC-SOD on threeconsecutive days. On the fourth day, the spleens were removed anddisintegrated. Spleen cells were fused with a mouse myeloma cell-lineaccording to standard techniques (St. Groth and Scheidegger, J. Immunol.Methods 35, 1980, pp. 1-21). Clones producing anti-EC-SOD wereidentified by means of an ELISA technique (Pouillard and Hoffman,Methods in Enzymology, 92, 1983, pp. 168) and further subcloned.Finally, two clones, "14,B7" and "6,H6" were selected for antibodypreparation on a larger scale by means of culture in the abdominalcavity of mice. Antibodies were then isolated from the ascites fluid byadsorption on and desorption from Protein A-Sepharose® as recommended bythe manufacturer (Pharmacia, Uppsala, Sweden). The elution from thecolumn was performed with 0.1M glycine-HCl pH 3.0. Immediately afterelution, the pooled IgG were titrated to pH 7.0 and then dialyzedagainst 50 mM Tris-HCl pH 8.0+0.15M NaCl , 0.02% NaN₃.

EXAMPLE 16 Immobilization of monoclonal anti-EC-SOD on CNB3-activatedSephorose®

Since the "6.H6" monoclonal antibody was found to bind n-EC-SOD verystrongly Kd<•10¹² M), the "14B7" antibody (Kd˜10⁶ M) was selected forEC-SOD purification purposes. Prior to the coupling to CNBr activatedSepharose, the azide in the IgG-solution (cf. Example 15) had to beremoved and the buffer had to be changed to a "coupling buffer" (=0.1MNa carbonate pH 8.3+0.5M NaCl). This was performed with a PD10 column bythe procedure recommended by the manufacturer (Pharmacia, Uppsala,Sweden). The CNBr-activated Sepharose® was swollen and prepared asrecommended by the manufacturer (Pharmacia, Uppsala, Sweden). TheCNBr-activated Sepharose® was then added to the IgG-solution (incoupling buffer) in an amount predicted to produce a coupling density ofabout 2 mg of IgG per ml of gel. The mixture was incubated at roomtemperature for about 2 hours on a "shaker". The buffer was then removedfrom the gel and analyzed for remaining protein. Over 97% coupling wasachieved. Remaining active groups on the IgG-coupled gel were thenblocked by incubation with 1M ethanolamine at pH 9.3 in 2 hours at roomtemperature. Excess of ethanolamine and adsorbed protein was finallywashed away with alternately "coupling buffer" (see above) and 0.1MNa-acetate, pH 4.0+0.5M NaCl, four to five times. The gel was stored in50 mM potassium phosphate, pH 7.4+0.5M NaCl+0.02% NaN₃ ⁻. The maximumbinding capacity of the monoclonal IgG-Sepharose® was determined byincubation for 3 hours with an excess of purified EC-SOD (Example 4-6)and subsequent analysis of remaining EC-SOD activity in the supernatantafter centrifugation. The result was compared with the analogousincubation with Sepharose® 4B.

To 1 ml of EC-SOD in "coupling buffer", 10, 50, 100 and 1000 μl of a 50%suspension of the monoclonal anti-EC-SOD-Sepharose® were added. Aparallel incubation in the same buffer of a 80% suspension of Sepharose®HB was performed. The solutions were incubated at room temperature in 3hours. After centrifugation the remaining EC-SOD activities in thesupernatants were determined. Using the resulting figures it could becalculated that the EC-SOD binding capacity of the gel was about ˜6000units of EC-SOD per ml of 50% gel suspension (=˜12000 U/ml of gel). Thisfigure is equal to about 6% of the theoretical maximum binding capacityand is close to what is generally achieved with randomly coupled IgG.

EXAMPLE17 Isolation of recombinant EC-SOD staring with monoclonalanti-EC-SOD Sepharose®

The entire purification procedure was performed at +4° C. About 5 litersof medium from cultures of CHO-K1/pPSneo-18 cells, containing about 300U EC-SOD activity (˜2.6 μg) per ml, were centrifuged to remove anycellular debris and precipitates. To bind the EC-SOD in the medium(˜1,500,000 units), about 125 ml of monoclonal anti-EC-SOD-Sepharose®was used (Example 16). The IgG-Sepharose was packed in a chromatographycolumn with a diameter of 5 cm and a height of about 6.5 cm. The columnwas washed with 50 mM sodium phosphate +0.5M NaCl, pH 7.0, prior to"sample application". The culture medium was applied with a rate of 100ml/h and the absorbance of 280 nm was monitored . Proteins loosely boundto the IgG-Sepharose were eluted with 50 mM sodium phosphate, pH6.5+0.5M NaCl. The elution continued until a very low AD₂₈₀ wasattained. The column was then washed with 650 ml of 50 mM AMP(=1-aminomethylpropanol)-HCl, pH 9.0 and the EC-SOD was eluted with ˜1liter of 50 mM AMP-HCl, pH 9.0+1M KSCN. The elution rate was 100 ml/h.EC-SOD-activity and absorbance at 280 nm were analyzed and plottedversus elution volume (FIG. 12). Remaining absorbance at 280 nm afterthe protein peak originates from the KSCN. The EC-SOD activity peak wasthen pooled. To reduce the ionic strength and to optimize the binding ofthe enzyme for the ion exchange gel, in the following step, the pool wasdiluted with about 14 volumes of distilled water and finally titrated topH 8.5 with 2M AMP. The recovery in the IgG-affinity step was 60%.

About 10 ml of DEAE-Sephacel® was washed with 50 mM sodium phosphate, pH6.5 and packed in a chromatography column with a diameter of 5 cm to aheight of about 0.5 cm. The diluted EC-SOD pool from the IgG-column wasallowed to adsorb to the DEAE-Sephacel® by pumping the pool through thecolumn with a rate of 60 ml/h. The column was then washed with 50 mMsodium phosphate, pH 8.5 until the absorbance at 280 nm was close tozero. The enzyme was eluted with 50 mM sodium phosphate, pH 8.5+0.25MNaCl. The absorbance at 280 nm and the EC-SOD. activity was analyzed andplotted versus elution volume (FIG. 13). The peak fractions were pooled,dialyzed against 50 mM sodium phosphate pH 6.5 and concentrated to about6 ml. The recovery in this step was about 100%.

The EC-SOD was finally purified by adsorption/desorption onheparin-Sepharose®. 12 ml of heparin-Sepharose® were prepared asrecommended by the manufacturer (Pharmacia, Uppsala, Sweden) and washedwith 50 mM sodium phosphate pH 6.5+1M NaCl and then with 50 mM sodiumphosphate pH 6.5. The heparin-Sepharose® gel was packed in achromatography column with a diameter of 2.5 cm (height about 2.5 cm).The concentrated and dialyzed pool from the DEAE-Sephacel® column (6 mlwith an EC-SOD-activity of about 185,000 U/ml, was applied on the columnwith an elution rate of 10 ml/h. The absorbance at 280 nm was monitored.The elution was started with 50 mM sodium phosphate pH 6.5 and enelution rate of 20 ml/h. When the absorbance at 280 nm approached thebaseline, the EC-SOD was eluted with a NaCl gradient from 0M to 1M NaCl.The gradient volume was 270 ml. To protect the EC-SOD from the high NaClconcentration, the fractions were diluted (from the start of thegradient) with distilled water. A T-pipe was inserted into the plastictube from the eluting end of the column, and distilled water was pumpedinto the eluting fluid at twice the column elution rate.

The EC-SOD-activity eluted in one peak (FIG. 14) at about the same NaClconcentration as the the C peak in Example 6. The center of the peak waspooled (Pool 1). The specific activity of Pool I (U/ml divided by AD₂₈₀)was 88,400. The specific activity of native EC-SOD prepared fromumbilical cord homogenate (cf. Example 1) by the same procedure was88,200. These figures are therefore almost identical and somewhat higherthan previously published (Marklund, Proc. Natl. Acad. Sci. USA 79,1982, pp. 7634-7638). These two preparations were analyzed in Examples12-14 and 18-20. The sides of the peak were also pooled (Pool II). Thepools were concentrated and dialyzed against 50 mM sodium phosphate, pH6.6. The yield of EC-SOD was about 60% in the heparin-Sepharose® step.The pools contained 600,000 U (about 5.3 mg) in all which is 40% of theoriginal activity in the CHO-K1/pPS3neo-18 culture medium.

EXAMPLE 18 Determination of molecular size of native EC-SOD andrecombinant EC-SOD by means of gel chromatography

The molecular weight of the native enzyme from umbilical cord and therecombinant enzyme was estimated by means of gel filtration on aSephacryl S-300® column. The column (1.6 cm in diameter, length 96 cm)was eluted with 10 mM potassium phosphate, pH 7.4+0.15M NaCl as eluent.The column was calibrated with ferritin (440,000), IgG (150,000), bovineserum albumin (67,000), ovalbumin (43,000), chymotrypsinogen (25,000),and ribonuclease (13,700) (molecular weights in parenthesis).

Native and recombinant EC-SOD eluted from the column at positionscorresponding to molecular weights of 136,000 and 151,000 respectively.The recombinant EC-SOD thus appeared to be slightly larger. Part of thedifference may be due to the partial degradation of the subunits of thenative enzyme as seen in the SDS-PAGE experiments below (Example 19).The heterogenicity of native EC-SOD upon chromatography on wheat germlectin (Example 12) also points to partial degradation of thecarbohydrate part of the enzymes.

EXAMPLE19 Analysis of native umbilical cord EC-SOD and recombinantEC-SOD by means of electrophoresis in gradient polyacrylamide gels inthe presence of sodium dodecyl sulphate

The molecular weight of the subunits of native- and recombinant EC-SODwas compared by electrophoresis in gradient gels (10-15% in the presenceof SDS.

Approximately 25 μg, of each enzyme (n-EC-SOD and r-EC-SOD) werefreeze-dried and thin dissolved in 50 μg of a sample mixture containing5% sucrose, 5 mM EDTA, 5% 2-mercaptoethanol and 2% SDS in a buffercomposed of 0.4M boric acid and 0.41M Tris, pH 8.64. The samples wereboiled for 5 minutes and immediately cooled on ice. About 1 μl (about0.2 μg) of each sample was applied on a Pharmacia Phast System gradient(10-15%) polyacrylamide gel and then run on a Pharmacia Phast SystemInstrument as recommended by the manufacturer (Pharmacia, Uppsala,Sweden in Phast System™ Separation Technique File No. 110). Theresulting gel was stained with Coomassie brilliant blue, cf. FIG. 15.From left to right the lanes contain recombinant EC-SOD, nativeumbilical cord EC-SOD and a mixture of molecular weight markers (94,000,67000, 43,000, 29,006, 20,100 and 14,400). The marker with a mobilitysimilar to the EC-SOD's is 29,000 in molecular weight. No impuritiescould be detected in the EC-SOD's. Recombinant EC-SOD has one band witha molecular weight of about 32,000. Native EC-SOD shows two bands, thelarger with apparently the same molecular weight as recombinant EC-SODand the smaller with a molecular weight of about 28,000. The relativeamounts of the two bands vary from preparation to preparation of nativeEC-SOD and the heterogeneity is probably due to partial degradation ofthe enzyme.

EXAMPLE 20 Comparison between the amino acid composition of native andrecombinant EC-SOD and the amino acid sequence deduced from the cDNAsequence encoding EC-SOD

The amino acid composition of native umbilical cord ECSOD andrecombinant EC-SOD is shown in Table II. Tryptophan was not included inthe comparison since it cannot be reliably obtained in an amino acidanalyser. It appears from the Table that the native and recombinantenzymes are almost identical in composition. The agreement with thefigures deduced from the cDNA sequence is also very good. The resultsindicate that the native and recombinant enzymes are virtually identicaland that the amino acid sequence deduced from the cDNA sequence iscorrect.

                  TABLE II                                                        ______________________________________                                        Amino acid composition of human EC-SOD                                        % residues/total residues                                                                        native    recombinant                                      from DNA sequence  enzyme    enzyme                                           Amino acid                                                                            +Trp     -Trp      -Trp    -Trp                                       ______________________________________                                        Phe     3.2      3.2       3.3     3.2                                        Leu     6.3      6.5       6.8     6.7                                        Ile     1.8      1.8       1.6     1.4                                        Met     0.9      0.9       1.2     0.9                                        Val     7.7      7.8       6.4     6.2                                        Ser     6.8      6.9       6.8     7.8                                        Pro     5.9      6.0       6.1     6.2                                        Thr     3.2      3.2       2.9     3.4                                        Ala     12.2     12.4      13.1    12.5                                       Tyr     1.4      1.4       1.5     1.4                                        His     4.1      4.1       4.2     3.9                                        Gln     5.0      5.1       --      --                                         Glu     6.8      6.9       --      --                                         Glx     11.7     12.0      11.7    12.1                                       Asn     3.2      3.2       --      --                                         Asp     5.9      6.0       --      --                                         Asx     9.0      9.2       9.3     9.3                                        Lys     2.3      2.3       1.9     2.5                                        Cys     2.7      2.8       2.8     2.8                                        Trp     2.3      --        --      --                                         Arg     9.0      9.2       9.8     9.4                                        Gly     9.9      10.1      10.5    10.4                                       ______________________________________                                    

We claim:
 1. A recombinant DNA molecule comprising DNA coding for anEC-SOD-like polypeptide comprising an amino acid sequence different frombut substantially corresponding to the amino acid sequence of maturehuman extracellular superoxide dismutase (EC-SOD) type C as given inFIGS. 5a and 5b, whereby said polypeptide exhibits superoxidedismutasing and heparin binding activities, and is bound by an antibodywhich binds human EC-SOD type C but does not bind human CuZnSOD.
 2. Ahost cell transformed with the molecule of claim 1, said DNA beingoperably linked with a promoter functional in the host cell.
 3. A methodof producing an EC-SOD-like polypeptide which comprises cultivating thehost cell of claim 2 under conditions conductive to expression of saidDNA and production of said EC-SOD-like polypeptide thereby, andrecovering said EC-SOD-like polypeptide.
 4. The molecule of claim 1,wherein the superoxide dismutasing activity of the polypeptide issubstantially reduced when the polypeptide is incubated with polyclonalantibody raised against human EC-SOD type C.
 5. The host cell of claim2, wherein the superoxide dismutasing activity of the polypeptide issubstantially reduced when the polypeptide is incubated with polyclonalantibody raised against human EC-SOD type C.
 6. The method of claim 3,wherein the superoxide dismutasing activity of the polypeptide issubstantially reduced when the polypeptide is incubated with polyclonalantibody raised against human EC-SOD type C.
 7. A recombinant DNAmolecule comprising DNA coding for an EC-SOD-like polypeptide comprisingan amino acid sequence different from but substantially corresponding tothe amino acid sequence of mature human EC-SOD type C as given in FIGS.5a and 5b, whereby said polypeptide exhibits superoxide dismutasingactivity, said activity being substantially reduced when the polypeptideis incubated with a polyclonal antibody raised against human EC-SOD typeC.
 8. A host cell transformed with the molecule of claim 7, said DNAbeing operably linked with a promoter functional in the host cell.
 9. Amethod of producing an EC-SOD-like polypeptide which comprisescultivating the host cell of claim 8 under conditions conductive toexpression of said DNA and production of said EC-SOD-like polypeptidethereby, and recovering said EC-SOD-like polypeptide.
 10. A recombinantDNA molecule comprising isolated or synthetic DNA coding for apolypeptide having superoxide dismutasing activity, said DNA beingselected from the group consisting of:a) a DNA which is sufficientlysimilar to the cDNA sequence of mature human EC-SOD type C as set forthin FIGS. 5a and 5b, so that one strand of said DNA hybridizes to astrand of said cDNA when incubated for 18 hours at 41° C. in a solutioncomprising 20% formamide, and washed in 0.2× SSC solution, and b) a DNAwhich encodes a polypeptide encoded by a DNA of (a) above,saidpolypeptide having superoxide dismutasing activity being more similar inamino acid sequence to human EC-SOD type C as set forth in FIGS. 5a and5b than to any naturally occurring CuZnSOD.
 11. A host cell transformedwith the molecule of claim 10, said DNA being operably linked with apromoter functional in the host cell.
 12. A method of producing anEC-SOD-like polypeptide which comprises cultivating the host cell ofclaim 11 under conditions conducive to expression of said DNA andproduction of said EC-SOD-like polypeptide thereby, and recovering saidEC-SOD-like polypeptide.
 13. A non-naturally-occurring EC-SOD-likepolypeptide comprising an amino acid sequence different from butsubstantially corresponding to the amino acid sequence of mature humanEC-SOD type C, whereby said polypeptide exhibits superoxide dismutasingand heparin binding activities, and is bound by an antibody which bindshuman EC-SOD type C but does not bind human CuZnSOD.
 14. An EC-SOD likepolypeptide, in essentially pure form as determined by SDS-PAGE, saidpolypeptide comprising an amino acid sequence substantiallycorresponding to the amino acid sequence of mature human EC-SOD type C,whereby said polypeptide exhibits superoxide dismutasing and heparinbinding activities, and is bound by an antibody which binds human EC-SODtype-C but does not bind human CuZnSOD.
 15. The EC-SOD-like polypeptideof claim 13, wherein the superoxide dismutasing activity of thepolypeptide is substantially reduced when the polypeptide is incubatedwith polyclonal antibody raised against human EC-SOD type C.
 16. TheEC-SOD-like polypeptide of claim 14, wherein the superoxide dismutasingactivity of the polypeptide is substantially reduced when thepolypeptide is incubated with polyclonal antibody raised against humanEC-SOD type C.
 17. A non-naturally occurring EC-SOD-like polypeptidecomprising an amino acid sequence different from but substantiallycorresponding to the amino acid sequence of mature human EC-SOD type C,whereby said polypeptide exhibits superoxide dismutasing activity, saidactivity being substantially reduced when the polypeptide is incubatedwith a polyclonal antibody raised against human EC-SOD type C, saidpolypeptide being bound by an antibody which binds human EC-SOD type Cbut does not bind human CuZnSOD.
 18. An EC-SOD like polypeptide, inessentially pure form as determined by SDS-PAGE, said polypeptidecomprising an amino acid sequence substantially corresponding to theamino acid sequence of mature human EC-SOD type C, whereby saidpolypeptide exhibits superoxide dismutasing activity, said activitybeing substantially reduced when the polypeptide is incubated with apolyclonal antibody raised against human EC-SOD type C, said polypeptidebeing bound by an antibody which binds human EC-SOD type C but does notbind human CuZnSOD.
 19. A non-naturally occurring EC-SOD-likepolypeptide, comprising an amino acid sequence different from that ofhuman EC-SOD type C, said polypeptide being encoded by a DNA which issufficiently similar to the cDNA sequence of mature human EC-SOD type Cas set forth in FIGS. 5a and 5b so that one strand of said DNAhybridizes to a strand of said cDNA, when incubated for 18 hours at 41°C. in a solution comprising 20% formamide, and washed in 0.2× SSCsolution, said polypeptide having superoxide dismutasing activity andbeing more similar in amino acid sequence to human EC-SOD type C as setforth in FIGS. 5a and 5b than to any normally occurring CuZnSOD.
 20. AnEC-SOD-like polypeptide, in essentially pure form as determined bySDS-PAGE, said polypeptide being encoded by a DNA which is sufficientlysimilar to the cDNA sequence of mature human EC-SOD type C as set forthin FIGS. 5a and 5b so that one strand of said DNA hybridizes to a strandof said cDNA, when incubated for 18 hours at 41° C. in a solutioncomprising 20% formamide, and washed in 0.2× SSC solution, saidpolypeptide having superoxide dismutasing activity and being moresimilar in amino acid sequence to human EC-SOD type C as set forth inFIGS. 5a and 5b than to any naturally occurring CuZnSOD.
 21. Apolypeptide comprising the amino acid sequence of mature human EC-SODtype C, in essentially pure form as determined by SDS-PAGE.